UBC Theses and Dissertations

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UBC Theses and Dissertations

Comparative karyotype analysis of the Pseudotsuga genus Colangeli, Anna Maria 1982

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COMPARATIVE KARYOTYPE ANALYSIS OF THE PSEUDOTSUGA GENUS by Anna M a r i a C o l a n g e l i B.Sc., U n i v e r s i t y of B r i t i s h C o l u m b i a , 1978 A T h e s i s S u b m i t t e d i n P a r t i a l F u l f i l l m e n t of t h e R e q u i r e m e n t s f o r The D e g r e e o f M a s t e r of S c i e n c e The F a c u l t y of G r a d u a t e S t u d i e s F a c u l t y of F o r e s t r y We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d The U n i v e r s i t y of B r i t i s h C o l u m b i a A p r i l , 1982 (c) Anna M a r i a C o l a n g e l i , 1982 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 l m e n t of t h e r e q u i r e m e n t f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y of B r i t i s h C o l u m b i a , I w i l l a g r e e t h a t t h e 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 s t u d y . I f u r t h e r a g r e e 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 p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t or by h i s ( h e r ) r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f 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 a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p artment of ^ W The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r , B.C. Canada Date H(\<ck 1 Q frrQ. i i ABSTRACT N u m e r i c a l d a t a were c o l l e c t e d from t h e k a r y o t y p e s of s e v e n s p e c i e s i n t h e P s e u d o t s u g a genus, P^ f o r r e s t i i , P. s i n e n s i s , P. g a u s s e n i i , P. j a p o n i c a , P. w i l s o n i a n a , P. m a c r o c a r p a and b o t h v a r i e t i e s of P_^  m e n z i e s i i , ( D o u g l a s - f i r ) , m e n z i e s i i and g l a u c a . A c o m p a r a t i v e k a r y o t y p e s t u d y was made u t i l i z i n g chromosome number, s e c o n d a r y c o n s t r i c t i o n s and chromosomal c h a r a c t e r i s t i c s , t o i d e n t i f y and s e p a r a t e t h e d i f f e r e n t s p e c i e s . B o t h v a r i e t i e s of D o u g l a s - f i r c o n t a i n e d a s o m a t i c chromosome number of 2n=26 c o n s i s t i n g o f f i v e m e t a c e n t r i c s , s i x s u b m e t a c e n t r i c s and two t e l o c e n t r i c p a i r s . P_^  m a c r o c a r p a and t h e A s i a t i c s p e c i e s c o n t a i n e d a s o m a t i c chromosome number of 2n=24 c o n s i s t i n g of s i x m e t a c e n t r i c and s i x s u b m e t a c e n t r i c p a i r s . D o u g l a s - f i r c o u l d be i d e n t i f i e d by chromosome number. An a n a l y s i s o f v a r i a n c e and a m u l t i v a r i a t e s t e p w i s e d i s c r i m i n a n t f u n c t i o n a n a l y s i s were employed t o t e s t t h e v a l i d i t y of u s i n g k a r y o t y p e d a t a s u c h as arm r a t i o , c e n t r o m e r e i n d e x , m o r p h o l o g i c a l i n d e x and r e l a t i v e l e n g t h f o r c h a r a c t e r i z i n g t h e c y t o g e n e t i c a l and t h e g e o g r a p h i c a l d i f f e r e n c e s f o r e a c h o f t h e n=12 s p e c i e s . Good d i s c r i m i n a t i o n was shown among P_;_ m a c r o c a r p a and t h e A s i a t i c s p e c i e s f o r b o t h t h e a n a l y s i s of v a r i a n c e and t h e m u l t i v a r i a t e a n a l y s i s . The d i s c r i m i n a t i o n among t h e A s i a t i c s p e c i e s was f u r t h e r i m p r o v e d when t h e w e l l s e p a r a t e d P. m a c r o c a r p a was o m i t t e d . A h i g h l e v e l of d i s c r i m i n a t i o n was o b s e r v e d between t h e two v a r i e t i e s o f D o u g l a s - f i r by b o t h . t h e t - t e s t and t h e . m u l t i v a r i a t e i i i a n a l y s i s . The m u l t i v a r i a t e a n a l y s i s was p a r t i c u l a r l y h e l p f u l i n s e l e c t i n g v a r i a b l e s t h a t b e s t s e p a r a t e d t h e t a x a . To my knowledge , t h e s e r e s u l t s p r o v i d e t h e f i r s t r e p o r t e d k a r y o t y p e n u m e r i c a l a n a l y s i s f o r t h e P s e u d o t s u g a genus a s w e l l as t h e f i r s t r e p o r t e d k a r y o t y p e a n a l y s i s f o r P^ _ g a u s s e n i i . i v TABLE OF CONTENTS Page A b s t r a c t i i T a b l e Of C o n t e n t s i v L i s t Of T a b l e s - v i i L i s t Of F i g u r e s v i i i A c k n owledgements i x 1. I n t r o d u c t i o n 1 2. L i t e r a t u r e Review 4 2.1. Number Of S p e c i e s 4 2.2. D i s t r i b u t i o n Of S p e c i e s 4 2.3. Review Of K a r y o t y p e s 7 3. M a t e r i a l s And Methods 9 3.1. R e s e a r c h M a t e r i a l 9 3.2. C y t o l o g i c a l T e c h n i q u e s . . .' 10 3.2.1. Sample P r e p a r a t i o n 10 3.2.2. P r e p a r a t i o n Of M i c r o S l i d e s And P r i n t s 11 3.2.3. Measurements And C a l c u l a t i o n s 12 3.2.4. S e c o n d a r y C o n s t r i c t i o n s 14 3.3. S t a t i s t i c a l A n a l y s i s . 15 3.3.1. T - t e s t 15 3.3.2. One Way A n a l y s i s Of V a r i a n c e 16 3.3.3. M u l t i v a r i a t e A n a l y s i s 18 4. R e s u l t s 20 4.1. Chromosomal C h a r a c t e r i s t i c s 20 4.1.1. P. m e n z i e s i i 20 4.1.1.1. C o a s t a l D o u g l a s - f i r 20 4.1.1.2. I n t e r i o r D o u g l a s - f i r 22 4.1.2. P. m a c r o c a r p a 25 4.1.3. P. f o r r e s t i i 29 4.1.4. P. s i n e n s i s •. 31 4.1.5. P. g a u s s e n i i 35 4.1.6. P. j a p o n i c a 38 4.1.7. P. w i l s o n i a n a 38 4.1.8. S e c o n d a r y C o n s t r i c t i o n s 42 4.2. C o m p a r i s o n 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 (n=13) by T - t e s t 45 4.3. C o m p a r i s o n o f t h e n=12 S p e c i e s by A n a l y s i s o f V a r i a n c e 49 4.3.1. Long Arm L e n g t h 49 4.3.2. S h o r t Arm L e n g t h 49 4.3.3. T o t a l L e n g t h ... 50 4.3.4. Arm R a t i o 54 4.3.5. C e n t r o m e r e Index 54 4.3.6. M o r p h o l o g i c a l Index 55 4.3.7. R e l a t i v e L e n g t h 59 4.4. C o m p a r i s o n of 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 ( n -13) by M u l t i v a r i a t e A n a l y s i s 59 4.5. C o m p a r i s o n o f t h e n=12 S p e c i e s by M u l t i v a r i a t e A n a l y s i s 61 5. D i s c u s s i o n 69 5.1. Chromosome Number 70 5.2. Chromosome M o r p h o l o g y 71 5.3. S e c o n d a r y C o n s t r i c t i o n s 72 5.4. C o a s t a l V e r s u s I n t e r i o r D o u g l a s - f i r (n=!3) .. . 75 v i 5.5. Variation Among The N=12 Species 77 ' 6. Summary 81 7. References 84 v i i L I S T OF TABLES 1 A n a l y s i s Of V a r i a n c e And E x p e c t e d Mean S q u a r e s 17 2 M o r p h o m e t r i c and Index V a l u e s o f C o a s t a l D o u g l a s - f i r .. 21 3 M o r p h o m e t r i c and Index V a l u e s of I n t e r i o r D o u g l a s - f i r . 24 4 M o r p h o m e t r i c and Index V a l u e s o f P_^  m a c r o c a r p a .• 27 5 M o r p h o m e t r i c and Index V a l u e s of f o r r e s t i i 30 6 M o r p h o m e t r i c and Index V a l u e s of P_^  s i n e n s i s 33 7 M o r p h o m e t r i c and Index V a l u e s of P_;_ g a u s s e n i i 36 8 M o r p h o m e t r i c and Index V a l u e s of P_;_ j a p o n i c a 39 9 M o r p h o m e t r i c and Index V a l u e s o f P_;_ w i l s o n i a n a 41 10 L o c a t i o n and F r e q u e n c y of S e c o n d a r y C o n s t r i c i t o n s 44 11 T - t e s t : C o a s t a l V e r s u s I n t e r i o r D o u g l a s - f i r 48 12 Duncans M u l t i p l e Range T e s t "Q" 51 13 Duncans M u l t i p l e Range T e s t "P" 52 14 Duncans M u l t i p l e Range T e s t "TL" 53 15 Duncans M u l t i p l e Range T e s t "AR" 56 16 Duncans M u l t i p l e Range T e s t " C I " 57 17 Duncans M u l t i p l e Range T e s t "MI" 58 18 Duncans M u l t i p l e Range T e s t "RL" 60 19 The D i s c r i m i n a n t A n a l y s i s of t h e D o u g l a s - f i r V a r i e t i e s (n=13) 62 20 The D i s c r i m i n a n t A n a l y s i s o f t h e s i x P s e u d o t s u g a s p e c i e s (n=12) 64 21 The D i s c r i m i n a n t A n a l y s i s of t h e f i v e A s i a t i c S p e c i e s . 67 v i i i L I S T OF FIGURES 1 D i s t r i b u t i o n o f t h e P s e u d o t s u g a Genus 5 2 Metaphase Chromosomes from C o a s t a l D o u g l a s - f i r 23 3 Metaphase Chromosomes from I n t e r i o r D o u g l a s - f i r 26 4 Metaphase Chromosomes from P_^  m a c r o c a r p a 28 5 Metaphase Chromosomes from P^ f o r r e s t i i 32 6 Metaphase Chromosomes from P_j_ s i n e n s i s 34 7 Me t a p h a s e Chromosomes from P_^  g a u s s e n i i 37 8 Me t a p h a s e Chromosomes from P^ j a p o n i c a 40 9 Metaphase Chromosomes from P_^  w i l s o n i a n a . 43 10 I d i o g r a m s o f t h e P s e u d o t s u g a S p e c i e s 47 11 H i s t o g r a m o f t h e C a n o n i c a l V a r i a b l e f o r D o u g l a s - f i r ... 63 12 P l o t o f t h e n=12 S p e c i e s Means 66 13 P l o t o f t h e F i v e A s i a t i c S p e c i e s Means 68 i x ACKNOWLEDGEMENTS I w i s h t o e x p r e s s my a p p r e c i a t i o n t o D r . 0. S z i k l a i , o f t h e F a c u l t y o f F o r e s t r y , under whose s u p e r v i s i o n t h i s s t u d y was u n d e r t a k e n , f o r h i s v a l u a b l e a d v i c e and c r i t i c i s m d u r i n g t h e r e s e a r c h , and f o r h i s g u i d a n c e i n t h e w r i t i n g o f t h i s r e p o r t . I am a l s o g r a t e f u l t o t h e o t h e r members o f my g r a d u a t e c o m m i t t e e , D r . K. C o l e , D r . Y.A. E l - K a s s a b y and Dr. D. L e s t e r f o r t h e i r keen i n t e r e s t i n my r e s e a r c h and f o r t h e i r r e v i e w o f t h i s r e p o r t . I am e s p e c i a l l y i n d e p t e d t o D r . Y.A. E l - K a s s a b y f o r h i s s u g g e s t i o n s and a s s i s t a n c e i n t h e s t a t i s t i c a l a n a l y s e s of t h e d a t a . W i t h o u t h i s c o n s t a n t m o r a l s u p p o r t and e n t h u s i a s m t h i s t h e s i s would n o t be a t t h e s t a g e i t i s t o d a y . I t h ank Mr. J . Emmanuel and Mr. B. Wong f o r t h e i r a s s i s t a n c e i n s e t t i n g up t h e computer programs and Mrs. E. F e e l e y f o r h e r t e c h n i c a l a s s i s t a n c e w i t h t h e p h o t o g r a p h y . F i n a n c i a l a s s i s t a n c e was g i v e n i n t h e form of F a c u l t y o f F o r e s t r y T e a c h i n g A s s i s t a n c e a s w e l l a s t h e D o n a l d S. McPhee F e l l o w s h i p Fund, G.S. A l l e n M e m o r i a l S c h o l a r s h i p , D r . G.S. A l l e n S c h o l a r s h i p i n F o r e s t G e n e t i c s and t h e Don B u c k l a n d M e m o r i a l S c h o l a r s h i p . To a l l t h o s e i n any way a s s o c i a t e d w i t h t h e s e f u n d s I e x t e n d my s i n c e r e a p p r e c i a t i o n . F i n a l l y I owe my d e e p e s t g r a t i t u d e t o my p a r e n t s f o r t h e i r c o n s t a n t encouragement d u r i n g my e n t i r e a c a d e m i c c a r e e r and t o my f i a n c e M i k e D e c k e r , who n o t o n l y s p e n t many weekends c o l l e c t i n g and p r e p a r i n g samples and l o n g h o u r s i n t h e d a r k r o o m d e v e l o p i n g f i l m but whose m o r a l s u p p o r t and e n c o u r a g e m e n t was a g r e a t a s s e t t o w a r d s t h e c o m p l e t i o n of t h i s t h e s i s . 1 1. INTRODUCTION The genus P s e u d o t s u g a , C a r r . a member o f t h e f a m i l y PINACEAE, has a r a n g e c o n f i n e d t o E a s t e r n A s i a a n d W e s t e r n N o r t h A m e r i c a . P s e u d o t s u g a means f a l s e T s u g a from t h e r e s e m b l a n c e t o t h a t genus. A l l members of t h e genus a r e d i s t i n g u i s h e d by woody c o n e s w i t h p e r s i s t e n t s c a l e s and p r o t r u d i n g , t h r e e p r o n g e d b r a c t s ; s p i n d l e s h a p e d buds and f i r - l i k e o r y e w - l i k e narrow l e a v e s ; w i n ged s e e d ; and r e s i n b l i s t e r s on smooth b a r k t h a t becomes f u r r o w e d and m a r b l e d w i t h c o r k l a y e r s a s i t d e v e l o p s . The l a r g e u nwinged p o l l e n g r a i n s and t h e p o l l i n a t i o n mechanism c l o s e l y r e s e m b l e L a r i x ( C h r i s t i a n s e n , 1972). I t s i n t o l e r a n c e t o shade, f i r e r e s i s t a n t m a r b l e d b a r k , wood anatomy and t h e a p p e a r a n c e o f i t s s e e d l i n g s , s e e d s and c o n e s a r e s t r i k i n g l y s i m i l a r t o l a r c h , -- enough t o s u g g e s t t h a t t h e genus L a r i x has c o n t r i b u t e d most t o i t s o r i g i n , i f n o t i t s d i r e c t a n c e s t r y , ( S i l e n , 1978). T h e r e has been some c o n t r o v e r s y o v e r t h e e x a c t number of s p e c i e s w i t h i n t h e genus, but i t i s p r e s e n t l y b e l i e v e d t o i n c l u d e e i g h t s p e c i e s , two N o r t h A m e r i c a n and s i x A s i a t i c s p e c i e s . The N o r t h A m e r i c a n s p e c i e s a r e Pj_ m a c r o c a r p a ( T o r r . ) Mayr and P. m e n z i e s i i ( M i r b . ) F r a n c o ( D o u g l a s - f i r ) . The l a t t e r i s f u r t h e r s u b d i v i d e d i n t o two v a r i e t i e s P_;_ m e n z i e s i i v a r . m e n z i e s i i , ( c o a s t a l D o u g l a s - f i r ) and IP^ m e n z i e s i i v a r . g l a u c a , ( i n t e r i o r D o u g l a s - f i r ) . The s i x A s i a t i c s p e c i e s i n c l u d e P_;_ f o r r e s t i i C r a i b , P. s i n e n s i s Do.de, P. g a u s s e n i i F l o u s , P^ w i . l s o n i a n a H a y a t a , 2 P. b r e v i f o l i a Cheng e t L.K. Fu and P_;_ j a p o n i c a ( S h i r a s . ) B e i s s n . D o u g l a s - f i r , w h i c h d o m i n a t e s t h e most p r o d u c t i v e f o r e s t l a n d o f W e s t e rn N o r t h A m e r i c a , i s one o f t h e most v a l u a b l e t i m b e r t r e e s w i t h more g e n e t i c r e s e a r c h b e i n g done on t h i s s p e c i e s t h a n any o t h e r i n t h e region.. P^ m a c r o c a r p a has a v e r y l i m i t e d r a n g e i n S o u t h e r n C a l i f o r n i a and i s o f e conomic i m p o r t a n c e o n l y i n t h a t a r e a . The A s i a t i c s p e c i e s have r e s t r i c t e d r a n g e s and a r e of l i t t l e e c o n o m i c v a l u e . S i x o f t h e e i g h t s p e c i e s i n c l u d i n g t h e two v a r i e t i e s o f P. m e n z i e s i i have p r e v i o u s l y been k a r y o t y p e d , (Sax and Sax, 1933; Zenke, 1953; B a r n e r and C h r i s t i a n s e n , 1962; C h r i s t i a n s e n , 1963; Thomas and C h i n g , 1968; D o e r k s e n and C h i n g , 1972; L i v i n g s t o n , 1971; D e V e s c o v i and S z i k l a i , 1975; Wochuk e t a l . , 1980). P. b r e v i f o l i a and P^ g a u s s e n i i a r e t h e e x c e p t i o n s . A n u m e r i c a l a n a l y s i s o f t h e k a r y o t y p e d a t a f r o m t h i s genus has not been r e p o r t e d . S a y l o r (1972) k a r y o t y p e d a l l t h e s p e c i e s o f t h e subgenus P i n u s and f o u n d t h a t t h e k a r y o t y p e s of s p e c i e s o f p i n e s were s i m i l a r but n o t i d e n t i c a l , and t h e d i f f e r e n c e s d e t e c t e d were f r e q u e n t l y s u b t l e r a t h e r t h a n s t r i k i n g l y o b v i o u s . T h e s e s u b t l e v a r i a t i o n s between s p e c i e s c o u l d be s t a t i s t i c a l l y d e t e c t e d i f t h e i r o c c u r r e n c e i s c o n s i s t e n t . B a s e d on t h i s i d e a , t h e o b j e c t i v e s of t h e s t u d y a r e as f o l l o w s : (1) To a n a l y z e c o m p a r a t i v e l y t h e k a r y o t y p e s o f t h e P s e u d o t s u g a g enus. 3 ( 2 ) To d e s c r i b e and t a b u l a t e by s p e c i e s t h e 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 o f e v e r y chromosome. (3) To d e t e r m i n e i f t h e s p e c i e s can be i d e n t i f i e d and s e p a r a t e d by s t a t i s t i c a l methods b a s e d on t h e s e 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 . (4) To d e t e r m i n e i 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 can be i d e n t i f i e d and s e p a r a t e d by s t a t i s t i c a l methods b a s e d on t h e s e 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 . 4 2. LITERATURE REVIEW 2.1. Number Of S p e c i e s T h e r e has been some u n c e r t a i n t y i n t h e l i t e r a t u r e a b o u t t h e e x a c t number o f A s i a t i c s p e c i e s w i t h i n t h e P s e u d o t s u g a genus. Chengde (1981) d e s c r i b e d f i v e s p e c i e s , P_^  s i n e n s i s, P. g a u s s e n i i , P. b r e v i f o l i a , and P_^  w i l s o n i a n a f r o m s o u t h e r n C h i n a and P. j a p o n i c a f r o m J a p a n . Bean (1976) c o n s i d e r e d P_;_ w i l s o n i a n a t o be c l o s e l y r e l a t e d t o and p o s s i b l y n o t d i s t i n c t from P. f o r r e s t i i . D a l l i m o r e and J a c k s o n ( 1 9 6 6 ) , c o n s i d e r e d P. f o r r e s t i i synomynous f o r P^ w i l s o n i a n a and P^ g a u s s e n i i . 2.2. D i s t r i b u t i o n Of S p e c i e s The f i v e P s e u d o t s u g a s p e c i e s f o u n d i n C h i n a have d i s t i n c t and n o n - o v e r l a p p i n g r a n g e s (Chengde, 1981). The N o r t h A m e r i c a n s p e c i e s a r e c o n f i n e d t o W e s t e r n N o r t h A m e r i c a , west o f t h e R o c k i e s ( L i t t l e , 1952). The e i g h t s p e c i e s ( F i g u r e 1) a r e d i s t r i b u t e d as f o l l o w s : P. m e n z i e s i i v a r . menz i es i i : C o a s t a l D o u g l a s - f i r . The range e x t e n d s from s o u t h w e s t e r n B.C. t h r o u g h w e s t e r n W a s h i n g t o n and Oregon t o c e n t r a l , c o a s t a l C a l i f o r n i a , e a s t i n t o t h e C a s c a d e and S i e r r a Nevada r a n g e s a t e l e v a t i o n s f r o m sea l e v e l t o 2500 m. FIGURE 1: A map showing the distribution of the Pseudotsuga species. 6 P. m e n z i e s i i v a r . g l a u c a : I n t e r i o r D o u g l a s - f i r . The range e x t e n d s f r o m c e n t r a l B.C. and s o u t h w e s t e r n A l b e r t a , s o u t h t h r o u g h t h e Rocky M o u n t a i n r a n g e t o n o r t h e r n and c e n t r a l M e x i c o , west t o e a s t e r n W a s h i n g t o n , Oregon and Nevada, e a s t t o c e n t r a l C o l o r a d o and New M e x i c o a t e l e v a t i o n s from 650 t o 4000 m. P. m a c r o c a r p a : B i g c o n e D o u g l a s - f i r grows on t h e r o c k y s l o p e s of t h e m o u n t a i n s i n s o u t h e r n C a l i f o r n i a , from t h e S a n t a I n e z M o u n t a i n s t o t h e s o u t h e r n b o r d e r o f C a l i f o r n i a a t 300 t o 2700 m a l t i t u d e . P. f o r r e s t i i : s i t u a t e d i n n o r t h w e s t Yunnan, s o u t h e a s t T i b e t and s o u t h w e s t S i c h a n a t an e l e v a t i o n of 2400 t o 3000'm. P. s i n e n s i s: w i d e l y s c a t t e r e d i n west Hupeh, n o r t h e a s t and s o u t h Hunan, n o r t h e a s t Kweichow and s o u t h e a s t S i c h u a n a t an e l e v a t i o n o f 800 t o 1200 m. I t o c c u r s a t 1500 t o 2800 m i n s o u t h e a s t S i c h u a n , c e n t r a l and n o r t h e a s t Yunnan. P. g a u s s e n i i : t h e r e a r e v e r y few of t h e s e t r e e s i n t h e n a t u r a l f o r e s t s ; t h e y a r e f o u n d i n s o u t h A n h u e i and west and s o u t h S h e k i a n g a t an e l e v a t i o n of 600 t o 1500 m. P. w i l s o n i a n a ; d i s t r i b u t e d i n t h e C e n t r a l S i e r r a on t h e i s l a n d of Taiwan a t 800 t o 1500 m a l t i t u d e . 7 P. b r e v i f o l i a : a v e r y r a r e t r e e d i s t r i b u t e d t h r o u g h s o u t h w e s t Kwangsi a t an e l e v a t i o n of 1250 m. P. j a p o n i c a : a r a r e t r e e c o n f i n e d t o t h e p r o v i n a n c e s of T o s a , K i i and Yamato i n s o u t h e a s t J a p a n a t an e l e v a t i o n of 300 t o 1000 m. 2.3. Review Of K a r y o t y p e s The f i r s t r e p o r t e d chromosome a n a l y s i s of D o u g l a s - f i r was made by Sax and Sax (1933) where a h a p l o i d number of n=13 was r e p o r t e d . D o u g l a s - f i r was u n i q u e b e c a u s e a l l o t h e r s p e c i e s k a r y o t y p e d i n t h e P i n a c e a e had a h a p l o i d number of n=12. T h e s e r e s u l t s were c o n f i r m e d by Zenke ( 1 9 5 3 ) , who r e p o r t e d 13 b i v a l e n t s a t d i a k i n e s i s o f m e i o s i s . B a r n e r and C h r i s t i a n s e n (1962) p r o d u c e d t h e - f i r s t p h o t o m i c r o g r a p h and i d i o g r a m o f t h e 26 s o m a t i c chromosomes of D o u g l a s - f i r . The c o a s t a l and i n t e r i o r v a r i e t i e s of D o u g l a s - f i r were o b s e r v e d t o c o n t a i n a h a p l o i d c o m p l i m e n t o f n=l3 ( L i v i n g s t o n , 1971; D e V e s c o v i and S z i k l a i , 1975). 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 ) , L i v i n g s t o n (1971) and Wochok e_t a l . (1980) r e p o r t e d t h e s o m a t i c chromosome s e t of D o u g l a s - f i r t o c o n s i s t of 5 p a i r s of m e t a c e n t r i c s , 6 p a i r s o f . s u b m e t a c e n t r i e s and 2 p a i r s of t e l o c e n t r i c s , w h i l e D e V e s c o v i and S z i k l a i (1975) r e f e r r e d t o t h e l a s t two p a i r s as s u b t e l o c e n t r i e s . The s o m a t i c chromosome number of P_j_ m a c r o c a r p a was r e p o r t e d as n=12 (Chr i s t a i n s e n , 1963). P_;_ w i l s o n i a n a (Thomas and C h i n g , 1968), P^ s i n e n s i s , P. f o r r e s t i i and P^ j a p o n i c a ( D o e r k s e n and 8 C h i n g , 1972), were a l l f o u n d t o have a h a p l o i d c o m p l i m e n t of n=12 d i v i d e d i n t o s i x p a i r s of m e t a c e n t r i c s and s i x p a i r s o f s u b m e t a c e n t r i c s , s i m i l a r t o t h e m a j o r i t y of t h e s p e c i e s i n t h e P i n a c e a e . The k a r y o t y p e s of P^ g a u s s e n i i and P^ b r e v i f o l i a have not been r e p o r t e d i n t h e l i t e r a t u r e . 9 3. MATERIALS AND METHODS 3.1. R e s e a r c h M a t e r i a 1 V e g e t a t i v e m a t e r i a l was c o l l e c t e d f r o m s e v e n s p e c i e s i n t h e P s e u d o t s u g a genus i n c l u d i n g t h e two v a r i e t i e s of P_;_ m e n z i e s i i . S e e d s , t e r m i n a l buds and s e e d l i n g r o o t t i p s were u s e d as t h e s o u r c e of m i t o t i c c e l l s . The type, of m a t e r i a l k a r y o t y p e d and i t s o r i g i n a r e d e s c r i b e d below: P. m e n z i e s i i : T e r m i n a l buds were c o l l e c t e d f r o m t r e e s of d i f f e r e n t p r o v i n a n c e s p l a n t e d a t t h e U.B.C. R e s e a r c h F o r e s t . T h e s e t r e e s o r i g i n a t e d f r o m s e e d c o l l e c t e d f r o m t h e i r n a t u r a l h a b i t a t i n 1966, 1968 and 1969 by t h e I n t e r n a t i o n a l U n i o n o f F o r e s t R e s e a r c h O r g a n i z a t i o n , - ( I U F R O ) . F o u r t o f i v e t r e e s from 13 d i f f e r e n t p r o v i n a n c e s were s a m p l e d . T h e s e i n c l u d e d s e v e n c o a s t a l D o u g l a s - f i r (CDf) and s i x i n t e r i o r D o u g l a s - f i r ( I D f ) p r o v i n a n c e s , e x t e n d i n g i n a range from c e n t r a l B.C. t o n o r t h e r n C a l i f o r n i a . P. m a c r o c a r p a (PM): T e r m i n a l buds were c o l l e c t e d from s i x 15-y e a r - o l d t r e e s l o c a t e d a t t h e U.B.C. R e s e a r c h F o r e s t . P. f o r r e s t i i ( P F ) : Buds were c o l l e c t e d f r o m f o u r 12 t o 1 4 - y e a r -o l d t r e e s f r o m t h e B.C. F o r e s t S e r v i c e ' s B r e e d i n g A r b o r e t u m a t Cowichan L a k e , B.C. 10 P. s i n e n s i s ( P S ) : Seeds were r e c e i v e d from L u i Hung-eh of t h e Department o f F o r e s t r y , C h e k i a n g F o r e s t r y C o l l e g e , C h e k i a n g C h i n a . The s e e d s were c o l l e c t e d i n Kweichow, C h i n a i n 1978. P. g a u s s e n i i ( P G ) : Root t i p s were c o l l e c t e d f r o m f o u r , t w o - y e a r -o l d s e e d l i n g s a t t h e B o t a n i c a l G a r d e n s , U.B.C. The s e e d l i n g s o r i g i n a t e d f r o m t h e A r b o r e t e u m o f t h e C h i n e s e Academy of F o r e s t r y , P e k i n g . P. j a p o n i c a ( P J ) : Seeds were r e c e i v e d from K i h a c h i r o Ohba from Mei P r e f e c t u r e , J a p a n . The s e e d s were c o l l e c t e d f r o m f o u r t r e e s l o c a t e d i n t h e N a t i o n a l F o r e s t o f t h e Owase D i s t r i c t F o r e s t O f f i c e . P. w i l s o n i a n a (PW): Seeds were r e c e i v e d f r o m B i n g Yen Yang of t h e B o t a n i c a l G a r d e n i n T a i p e i , T a i w a n . The seeds, o r i g i n a t e d from f i v e t r e e s i n a n a t u r a l s t a n d i n c e n t r a l T a i w a n . 3.2. C y t o l o g i c a l T e c h n i q u e s D u p l i c a t e s e t s of v e g e t a t i v e m a t e r i a l were s u b j e c t e d t o s e v e r a l t r e a t m e n t s t a g e s b e f o r e d a t a c o n c e r n i n g t h e k a r y o t y p e s was c o l l e c t e d . 3.2.1. Sample P r e p a r a t i o n Seeds o f P J , PS and PW were i m b i b e d i n t a p w a t e r a t room t e m p e r a t u r e f o r 24 h o u r s , t h e n g e r m i n a t e d on a J a c o b s e n G e r m i n a t o r ( Z e p h y r N.V., K o e l - e n L u c h t t e c h i e k Z o e t e r m e e r , H o l l a n d ) . When t h e r a d i c a l s a t t a i n e d a l e n g t h of 5 t o 10 mm, t h e 11 g e r m i n a n t s were t r a n s f e r r e d t o t a p w a t e r and m a i n t a i n e d a t 4°C o v e r n i g h t , f o l l o w i n g w h i c h t h e y were s o a k e d i n a .1% s o l u t i o n o f c o l c h i c i n e f o r t h r e e h o u r s a t room t e m p e r a t u r e . The r o o t t i p s e x c i s e d f r o m t h e PG s e e d l i n g s were s i m i l a r l y t r e a t e d . The m a t e r i a l was f i x e d i n F a r m e r s F i x a t i v e (3:1 a b s o l u t e a l c o h o l : g l a c i a l a c e t i c a c i d ) a t room t e m p e r a t u r e f o r 24 h o u r s t h e n s t o r e d i n 70% e t h a n o l , a t 4°C u n t i l needed. The t e r m i n a l buds of CDf, I D f , PF and PM were c u t i n h a l f l o n g i t u d i n a l l y , s o a k e d i n water a t 4°C o v e r n i g h t , t r a n s f e r r e d i n t o .1% c o l c h i c i n e f o r 3 h o u r s a t room t e m p e r a t u r e and f i x e d i n F a r m e r s f o r 24 h o u r s . T h i s m a t e r i a l was a l s o s t o r e d i n 70% e t h a n o l u n t i l r e q u i r e d f o r k a r y o t y p i n g . 3.2.2. P r e p a r a t i o n Of M i c r o SI i d e s And P r i n t s The t e r m i n a l 2-5 mm of t h e r a d i c a l s 1 were s n i p p e d o f f t h e g e r m i n a n t s and washed t o remove e x c e s s e t h a n o l . Time and t e m p e r a t u r e o f h y d r o l y s i s was f o u n d t o be t h e c r i t i c a l s t a g e i n a c h i e v i n g optimum s t a i n i n g l a t e r i n t h e p r o c e d u r e . The optimum h y d r o l y s i s t i m e and t e m p e r a t u r e was d e t e r m i n e d f o r r o o t t i p s and buds. Root t i p s were h y d r o l y z e d i n 1M HC1 a t 60°C f o r 12 m i n u t e s w h i l e buds were t r e a t e d a t t h e same c o n c e n t r a t i o n and t e m p e r a t u r e f o r e i g h t m i n u t e s . The m a t e r i a l was t r a n s f e r r e d t o l e u c o - b a s i c f u c h s i n and kept i n t h e d a r k , a t room t e m p e r a t u r e . A f t e r 1.5 h o u r s , e x c e s s s t a i n was removed from t h e t i s s u e s by r i n s i n g t h e m a t e r i a l i n t h r e e 10 m i n u t e c h a n g e s o f S 0 2 w a t e r . The d e e p l y s t a i n e d m a t e r i a l was s q u a s h e d i n a d r o p of 45% a c e t i c a c i d u s i n g a c o v e r s l i p , h e a t and p r e s s u r e t o s p r e a d t h e c e l l s . The s l i d e s were made -semi-permanent by s e a l i n g t h e edges 1 2 of t h e c o v e r s l i p t o t h e s l i d e w i t h r u b b e r cement. C e l l s w i t h w e l l s p r e a d chromosomes were p h o t o g r a p h e d a t a m a g n i f i c a t i o n o f 1000X u s i n g an "Orthomat P h o t o U n i t " mounted on a L e i t z O r t h o l u x m i c r o s c o p e e m p l o y i n g Kodak T e c h n i c a l Pan 2415 f i l m . S i x t y t o one h u n d r e d d i f f e r e n t chromosome s p r e a d s were p h o t o g r a p h e d from e a c h s p e c i e s i n o r d e r t o o b t a i n 40 w e l l s p r e a d , unambiguous images w h i c h were t h e n measured. 3.2.3. Measurements And C a l c u l a t i o n s Due t o t h e s m a l l amount o f m a t e r i a l a v a i l a b l e from PF buds, o n l y 20 chromosome s p r e a d s c o u l d be u s e d . The n e g a t i v e s o f t h e 40(20) s p r e a d s were p r o j e c t e d o n t o a m i r r o r p l a c e d a t a 45 d e g r e e a n g l e , a p p r o x i m a t e l y 1.2 m i n f r o n t o f t h e p r o j e c t o r . The image was r e f l e c t e d up t o a h o r i z o n t a l s h e e t o f g l a s s a p p r o x i m a t e l y 30 cm above t h e c e n t e r o f t h e m i r r o r . The 1.2 m d i s t a n c e between t h e m i r r o r and p r o j e c t o r p r o v i d e d a f i n a l m a g n i f i c a t i o n o f 3700x. By p l a c i n g a s h e e t o f t r a c i n g p a p e r on t h e g l a s s t h e image of t h e n e g a t i v e was v i s i b l e . The chromosomes i n c l u d i n g c e n t r o m e r e s and s e c o n d a r y c o n s t r i c t i o n s were t r a c e d o n t o t h e p a p e r . A f t e r t h e chromosomes were t r a c e d , a l i n e was drawn down t h e m i d d l e o f e a c h chromosome, p a s s i n g t h r o u g h t h e • c e n t r o m e r e and r u n n i n g p a r a l l e l t o t h e s i d e s of t h e chromosome. The chromosomes of a g i v e n s p r e a d were a r b i t r a r i l y numbered 1-24 (1-26 f o r D o u g - l a s - f i r ) . The l e n g t h of e a c h arm was m e a s u r e d by r u n n i n g a map m e a s u r e r a l o n g t h e c e n t e r l i n e . The map m e a s u r e r was c a l i b r a t e d i n i n c r e m e n t s of 1/32 i n c h e s (.77 mm). T h e s e were 1 3 a r b i t r a r i l y l a b e l l e d map measure u n i t s . The s h o r t arm ( P ) , l o n g arm (Q) and t o t a l l e n g t h s (TL) were measured f o r e a c h chromosome. U s i n g t h e l e n g t h and t h e p o s i t i o n of t h e c e n t r o m e r e , homologous chromosomes were p a i r e d . The chromosome p a i r s were r a n k e d i n o r d e r of d e c r e a s i n g l e n g t h . F o r e a c h p a i r t h e m o r p h o m e t r i c v a l u e s Q, P and TL were a v e r a g e d and t h e f o l l o w i n g i n d e x v a l u e s were c a l c u l a t e d . Arm R a t i o , ( AR). T h i s v a l u e i s b a s e d on l o n g arm l e n g t h (Q) d i v i d e d by s h o r t arm l e n g t h ( P ) . C e n t r o m e r e Index, ( C I ) . The f o l l o w i n g f o r m u l a ( S t e p h e n s o n e t a l . , 1972) c a l c u l a t e s t h e r e l a t i o n s h i p between t h e s h o r t arm (P) and t h e t o t a l l e n g t h ( T L ) . CI = 100P/(P + Q) M o r p h o l o g i c a l Index, ( M I ) . T h i s v a l u e i s a p r o d u c t of t h e arm r a t i o and t h e t o t a l l e n g t h . ( G i a n n e l l i and H o w l e t t 1967). MI = ( P / Q ) ( P + Q) 1 4 R e l a t i v e L e n g t h , ( R L ) . R e l a t i v e l e n g t h i s d e f i n e d as t h e t o t a l l e n g t h of an i n d i v i d u a l chromosome i n a s i n g l e c e l l d i v i d e d by t h e a v e r a g e chromosome l e n g t h o f t h a t c e l l m u l t i p l i e d by 100. RL = T L / a v e r a g e TL x 100 The chromosomes were s e p a r a t e d i n t o d i f f e r e n t m o r p h o l o g i c a l g r o u p s b a s e d on t h e i r arm r a t i o a c c o r d i n g t o t h e c l a s s i f i c a t i o n s y s t e m of L e v a n e_t a l . ( 1 9 6 4 ) . Arm r a t i o s of 1.0 t o 1.7 a r e m e t a c e n t r i c s , 1.7 t o 3.0 a r e s u b m e t a c e n t r i c s , 3.0 t o 7.0 a r e a c r o c e n t r i c s and chromosomes w i t h t e r m i n a l c e n t r o m e r e s (arm r a t i o e q u a l s i n f i n i t y ) a r e t e l o c e n t r i c s . An i d i o g r a m was c o n s t r u c t e d w i t h t h e chromosomes a r r a n g e d i n d e c r e a s i n g l e n g t h w i t h t h e c e n t r o m e r e s a l o n g a h o r i z o n t a l l i n e and t h e s h o r t arms (P) up. 3.2.4. S e c o n d a r y C o n s t r i c t i o n s S e c o n d a r y c o n s t r i c t i o n s (SC) a p p e a r as e i t h e r d i s t i n c t g aps, d e f i n i t e b u t n o t c o m p l e t e c o n s t r i c t i o n s o r l i g h t l y s t a i n e d bands on t h e chromosomes. The p o s i t i o n of e v e r y s e c o n d a r y c o n s t r i c t i o n , whether a d i s t i n c t gap o r l i g h t l y s t a i n e d band and t h e chromosome arm on w h i c h i t o c c u r r e d was n o t e d . The c o n s t r i c t i o n s were i d e n t i f i e d by t h e f o l l o w i n g c o d e , u t i l i z i n g t h e r e f e r e n c e s y s t e m of P e d e r i c k ( 1 9 6 7 ) : - t h e chromosome on w h i c h i t was l o c a t e d (1 -13) , -whether l o n g o r s h o r t arm was i n v o l v e d , - p o s i t i o n of t h a t arm, ( d e n o t e d by p e r c e n t d i s t a n c e f f o m t h e c e n t r o m e r e ) . 15 F o r example, 4 L 54 would e x p r e s s a s e c o n d a r y c o n s t r i c t i o n on t h e l o n g arm of chromosome 4, a t a d i s t a n c e o f 54% t h e l e n g t h of t h e arm from t h e c e n t r o m e r e . To d i f f e r e n t i a t e between c o n s i s t e n t SC and a r t i f a c t s due t o uneven s t a i n i n g o r t r e a t m e n t p r o c e d u r e s , o n l y c o n s t r i c t i o n s t h a t were o b s e r v e d i n g r e a t e r t h a n o r e q u a l t o 70% o f t h e c e l l s were d e s i g n a t e d a s SC. 3.3. S t a t i s t i c a l A n a l y s i s T h r e e s t a t i s t i c a l methods were u t i l i z e d t o a n a l y z e t h e a v a i l a b l e d a t a c o m p i l e d on t h e s e v e n s p e c i e s . T h e s e i n c l u d e d t h e t - t e s t , one-way a n a l y s i s o f v a r i a n c e ( S t e e l and T o r r i e , 1980) and m u l t i v a r i a t e a n a l y s i s ( J e n n r i c h and Sampson, 1981; F a l k e n h a g e n and Nash, 1978) . 3.3.1. T - t e s t I t i s a w e l l e s t a b l i s h e d f a c t t h a t D o u g l a s - f i r has an e x t r a p a i r of chromosomes r e l a t i v e t o t h e o t h e r P s e u d o t s u g a s p e c i e s . S i n c e i t can be u n a m b i g u o u s l y i d e n t i f i e d by chromosome number, D o u g l a s - f i r was not i n c l u d e d i n a s t a t i s t i c a l a n a l y s i s w i t h t h e o t h e r s p e c i e - s . The m o r p h o m e t r i c and i n d e x v a l u e s of t h e two v a r i e t i e s of D o u g l a s - f i r were compared by t - t e s t s ( S t e e l and T o r r i e , 1980). The m o r p h o m e t r i c and i n d e x v a l u e s o f PG w i l l be compared t o t h e s p e c i e s c o n t a i n i n g t h e same chromosome numbers. 1 6 3.3.2. One Way A n a l y s i s Of V a r i a n c e An a n a l y s i s of v a r i a n c e was p e r f o r m e d on e a c h chromosome c h a r a c t e r i s t i c of t h e s p e c i e s c o n t a i n i n g an n-number o f 12. The a n a l y s i s was a c c o m p l i s h e d u s i n g ANOVAR, a computer p a c k a g e a v a i l a b l e f r o m t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a ' s Computing C e n t r e . W i t h e a c h s p e c i e s c o n s i s t i n g of 12 chromosomes and e a c h chromosome c o n t a i n i n g s e v e n v a r i a b l e s , 84 a n a l y s e s o f v a r i a n c e were p e r f o r m e d . Whenever a s i g n i f i c a n t F r a t i o was o b t a i n e d a Duncan's M u l t i p l e Range T e s t f o r u n e q u a l g r o u p s i z e s ( S t e e l and T o r r i e , 1980) was u s e d t o l o c a t e s i g n i f i c a n t d i f f e r e n c e s w i t h i n t h e g r o u p s o f measurement means. The ANOVA t a b l e and t h e E x p e c t e d Mean S q u a r e s b a s e d on t h e f o l l o w i n g l i n e a r model i s ' p r e s e n t e d i n T a b l e 1 . 17 y.. = n + s. + c,.,. Where: Y.. = measurement of the j**1 variable i n the 1 D .th l species, H = means of a l l observations over a l l chromosomes, species, th S. = effect of the l species, l : ( i ) j C,.^A = effect of the j*"*1 variable within the .th l species. TABLE 1 Analysis of Variance and Expected Mean Squares Source of Variation df Expected Mean Squares AMONG Species a - 1 2 2 a • + K<7 e s WITHIN Species (RESIDUAL) TOTAL a 2 n j a ~n 4 - a - 1 Where: a = n. = i K = number of n=12 species, number of observations per species, coefficient of the variance component, residual variance, = variation among species. 18 3.3.3. M u l t i v a r i a t e A n a l y s i s S t e p w i s e d i s c r i m i n a n t a n a l y s i s was p e r f o r m e d s e p a r a t e l y f o r D o u g l a s - f i r (n=l3) and t h e s p e c i e s c o n t a i n i n g an n-number of 12, u s i n g t h e computer program BMD:P7M ( J e n n r i c h and Sampson, 1981). The aim o f t h e a n a l y s i s i s t o f i n d l i n e a r c o m b i n a t i o n s of t h e o r i g i n a l d a t a but w i t h c o e f f i c i e n t s d e r i v e d so t h a t t h e peak a x i s shows t h e g r e a t e s t v a r i a t i o n among t h e d i f f e r e n t s p e c i e s . The s t e p w i s e p r o c e d u r e w i l l i n t r o d u c e one v a r i a b l e a t a t i m e a c c o r d i n g t o w h i c h one m a x i m i z e s t h e o v e r a l l m u l t i v a r i a t e F - r a t i o f o r t h e t e s t o f d i f f e r e n c e s among t h e s p e c i e s means. T h i s n o t o n l y i d e n t i f i e s t h e more i m p o r t a n t v a r i a b l e s i n t h e d i s c r i m i n a t i o n , but a l s o g i v e s t h e b e s t s u b s e t o f one, two, t h r e e o r more v a r i a b l e s f o r a f u r t h e r t e s t t o f i n d t h e optimum number of v a r i a b l e s t o i n c l u d e . The a n a l y s i s i s d i v i d e d c o n c e p t u a l l y i n t o two s t e p s : F i r s t , t h e d i f f e r e n t v a r i a t e s a r e t r a n s f o r m e d i n s u c h a way t h a t t h e swarm o f p o i n t s from e a c h sample becomes s p h e r i c a l i n s h a p e . T h i s s p h e r i c a l shape i m p l i e s t h a t t h e w i t h i n -g r o u p random e r r o r v a r i a t i o n has been s t a n d a r d i z e d so as t o be th e same i n e v e r y d i r e c t i o n . The swarm of sample means has been c h a n g e d i n shape and s i z e , but w i l l s t i l l be e l l i p s o i d a l . Second, t h e new c o o r d i n a t e a x e s a r e r o t a t e d so as t o be p a r a l l e l t o t h e p r i n c i p l e a x e s of t h e new e l l i p s o i d r e p r e s e n t i n g t h e swarm of sample means. The two s t e p s t o g e t h e r g i v e a s e t o f c a n o n i c a l v a r i a t e s , w h i c h not o n l y a r e u n c o r r e l a t e d but a l s o a r e u n a f f e c t e d by t h e u n i t s of measurement u s e d i n r e c o r d i n g t h e o r i g i n a l o b s e r v a t i o n s . The minimum F t o e n t e r and d e l e t e v a r i a b l e s was s e t 19 a t 2.50 t o p r e v e n t i n c l u s i o n of n o n - s i g n i f i c a n t v a r i a b l e s . J a c k -k n i f e d c l a s s i f i c a t i o n a n a l y s i s and p l o t s o f g r o u p means on t h e f i r s t t h r e e c a n o n i c a l v a r i a t e s were made. J a c k - k n i f i n g i s a p r o c e s s where t h e a n a l y s i s i s r e p e a t e d o m i t t i n g one sample a t a t i m e and c l a s s i f y i n g t h a t sample t o t h e g r o u p f o r w h i c h i t has t h e h i g h e s t p o s t e r i o r p r o b a b i l i t y of membership. The number of v a r i a b l e s u s e d were 91 f o r t h e n=13 s p e c i e s and 84 f o r t h e n=l2 s p e c i e s ( s e v e n v a r i a b l e s / c h r o m o s o m e ) . 20 4. RESULTS » 4.1. Chromosomal C h a r a c t e r i s t i e s A h a p l o i d chromosome number o f h=l2 was c o n s i s t e n t l y f o u n d i n a l l c e l l s a n a l y z e d f o r t h e A s i a t i c s p e c i e s (PF, PS, PG, PJ and PW) and t h e C a l i f o r n i a b i g c o n e D o u g l a s - f i r (PM). The h a p l o i d chromosome number of 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 was n=13. S e c o n d a r y c o n s t r i c t i o n s were o b s e r v e d i n many of t h e c e l l s m e a sured. The p r e s e n c e of t h e s e may be i m p o r t a n t i n i d e n t i f y i n g and s e p a r a t i n g s p e c i e s . 4.1.1. P^ m e n z i e s i i D o u g l a s - f i r d i f f e r e d f r o m t h e r e s t o f t h e P s e u d o t s u g a s p e c i e s by p o s s e s s i n g a h a p l o i d complement o f n=13 c o n s i s t i n g of f i v e m e t a c e n t r i c , s i x s u b m e t a c e n t r i c and two t e l o c e n t r i c p a i r s of chromosomes. 4.1.1.1. C o a s t a l D o u g l a s - f i r A c o m p l e t e summary o f t h e m o r p h o l o g i c a l d a t a f o r C o a s t a l D o u g l a s - f i r i s l i s t e d i n T a b l e 2. The s h o r t arm l e n g t h s ( P ) , t o t a l l e n g t h s (TL) and m o r p h o l o g i c a l i n d e x (MI) v a l u e s d i s p l a y a d e c r e a s i n g t r e n d f o r chromosomes 1-13. The l o n g arm l e n g t h s (Q) d e c r e a s e s f o r chromosomes 1-5, 6-11 and 12-13. The arm r a t i o (AR) and c e n t r o m e r e i n d e x (CI.) v a l u e s of t h e f i v e m e t a c e n t r i c chromosomes a r e s i m i l a r ( T a b l e 2 ) . AR v a l u e s r a n g e from 1.12 t o 1.14 w h i l e CI v a l u e s a r e between 46.9 -and TABLE 2: Morphometric and Index Values of Coastal Douglas-fir CHROMOSOME NUMBER LONG ARM MEAN SO CV SHORT ARM (P) MEAN SO CV TOTAL LENGTH (TL) MEAN SD CV ARM RATIO (AR) MEAN SD CV CENTROMERE INDEX (CI) MEAN SD CV MORPHOLOGICAL INDEX (MI) MEAN SO CV RELATIVE LENGTH (RL) MEAN SO CV 31.4 3.9 X8.8 27.9 5.8 20.0 59.3 11.3 19.1 1.13 .06 5.4 47.0 1.3 2.8 52.8 11.1 21.0 145 2.5 X.7 29.6 5.7 19.2 26.5 5.0 18.9 56.1 10.6 18.9 1.12 .05 4.2 47.2 .1.1 2.2 50.1 9.6 19.3 137 2.6 1.9 28.0 5.3 18.8 25.0 4.8 19.3 53.0 10.0 18.9 1.12 .05 4.6 47.2 1.2 2.6 47.5 9.5 20.0 129 2.9 2.3 27.1 S.2 19.2 24.0 4.7 19.7 51.0 9.8 19.0 1.14 .06 5.2 46.9 1.3 2.8 45.1 9.2 20.4 124 2.9 2.4 25.9 5.1 19.6 23.1 4.7 20.4 49.1 9.7 19.8 1.13 .05 5.5 47.1 1.3 2.8 43.8 9.2 21.0 120 3.0 2.5 27.5 5.4 19.7 12.7 2.2 17.1 40.2 7.5 18.7 2.16 .13 6.2 31.7 1.3 4.1 18.5 3.1 16.7 98 1.9 2.0 26.1 4.8 18.5 11.9 2.2 18.4 38.1 7.0 18.3 2.19 .09 4.1 31.4 .9 2.9 17.4 3.2 18.5 93 1.7 1.9 25.1 4.8 19.3 11.4 2.0 17.0 36.5 6.7 18.3 2.19 .15 6.7 31.4 1.5 4.6 16.7 2.8 16.9 89 2.2 2.5 23.9 4.4 18.5 10.8 2.0 18.3 34.7 6.4 18.3 2.23 .12 5.2 31.0 1.1 3.6 15.6 2.9 18.5 85 2.5 3.0 10 22.5 4.4 19.4 10.3 1.7 17.4 32.7 6.1 18.3 2.19 .13 4.5 31.4 1.3 4.1 14.9 2.7 17.0 80 2.7 3.4 11 20.9 4.1 19.6 9.6 1.8 18.6 30.5 5.8 19.0 2.18 .14 6.2 31.5 1.4 4.4 14.0 2.6 18.7 ' 74 2.5 3.4 12 28.6 5.4 18.7 0.0 28.6 5.3 18.7 8.00 0.0 0.0 — 70 2.2 3.2 13 23.0 4.4 18.9 0.0 23.0 4.3 18.9 8.00 0.0 0.0 56 2.0 3.5 to 22 47.2. The s u b m e t a c e n t r i c chromosomes a l s o have c o n s i s t e n t AR (2.16 t o 2.23) and CI (31.0 t o 31.7) v a l u e s . P o s s e s s i n g o n l y one arm, t h e t e l o c e n t r i c s (chromosomes 12 and 13) have an AR o f i n f i n i t y and CI and MI v a l u e s o f 0.00 ( T a b l e 2 ) . The maximum d i f f e r e n c e i n RL between n e i g h b o u r i n g chromosomes i s 22 u n i t s o c c u r r i n g between chromosome 5 ( m e t a c e n t r i c ) and chromosome 6 ( s u b m e t a c e n t r i c ) . The n e x t l a r g e s t d i f f e r e n c e (14 u n i t s ) o c c u r s between t h e t e l o c e n t r i c s (chromosomes 12 and 13). The r e m a i n i n g chromosomes have r e l a t i v e d i f f e r e n c e s o f 4 and 9 u n i t s between n e i g h b o u r i n g p a i r s . A p h o t o m i c r o g r a p h of t h e 26 metaphase chromosomes from t h e t e r m i n a l buds o f CDf i s p r e s e n t e d i n F i g u r e 2A. A k a r y o t y p e o f t h e 13 p a i r s o f chromosomes r a n k e d i n o r d e r of d e c r e a s i n g l e n g t h i s l o c a t e d i n F i g u r e 2B. 4.1.1.2. I n t e r i o r D o u g l a s - f i r The m o r p h o m e t r i c and i n d e x v a l u e s f o r I n t e r i o r D o u g l a s - f i r a r e summarized i n T a b l e 3. P and MI d i s p l a y d e c r e a s i n g t r e n d s f o r chromosomes 1-11. Q and TL v a l u e s e x h i b i t d e c r e a s i n g t r e n d s f o r chromosomes 1-5, 6-11 and 12-13. The AR v a l u e s f o r t h e chromsomes a r e q u i t e c o n s i s t e n t , w i t h t h e m e t a c e n t r i c s r a n g i n g between 1.11 and 1.15 and t h e s u b m e t a c e n t r i c s between 2.17 and 2 . 1 9 . The CI v a l u e s a r e a l s o s i m i l a r l y homogenous, r a n g i n g between 46.6 and 47.3 f o r t h e m e t a c e n t r i c s and 31.4 t o 31.6 f o r t h e s u b m e t a c e n t r i c s . The maximum d i f f e r e n c e i n RL between n e i g h b o u r i n g chromosomes (18 u n i t s ) i s f o u n d between chromosome 5 ( m e t a c e n t r i c ) and chromosome 6 ( s u b m e t a c e n t r i c ) . T h i s i s f o l l o w e d 23 8 ft «'» o 1 2 3 4 5 B (( is ii o n n n n 6 7 8 9 10 11 12 13 FIGURE 2: A) Metaphase chromosomes (2n=26) from a terminal bud c e l l of Coastal Douglas-fir, (2000x), B) Karyotype of the t h i r t e e n pai r s of CDf chromosomes (2000x). CHROMOSOME NUMBER 1 2 3 4 5 6 7 8 9 10 11 12 13 LONG ARM (Q) MEAN SD CV 3 6 . 3 7 . 4 2 0 . 3 3 4 . 0 6 . 8 1 9 . 9 3 2 . 4 6 . 4 1 9 . 6 3 1 . 2 6 . 3 2 0 . 3 2 9 . 9 6 . 2 2 0 . 6 3 2 . 6 7 . 0 2 1 . 4 3 0 . 2 6 . 4 2 1 . 3 2 8 . 6 6 . 2 2 1 . 7 2 7 . 0 5 . 8 2 1 . 5 2 5 . 0 5 . 0 1 9 . 9 2 3 . 3 4 . 6 1 9 . 7 3 1 . 8 6 . 3 1 9 . 9 2 5 . 1 5 .1 2 0 . 3 TABLE SHORT ARM (P) MEAN SD CV 3 2 . 5 6 . 8 2 0 . 9 3 0 . 7 6 .7 2 1 . 8 2 9 . 0 6 . 0 2 0 . 7 2 7 . 7 6 . 0 2 1 . 7 2 6 . 1 5 .7 2 1 . 7 1 4 . 9 2 . 9 1 9 . 6 1 3 . 9 3 .0 2 1 . 2 1 3 . 1 2 . 9 2 2 . 2 1 2 . 4 2 .6 2 1 . 0 1 1 . 5 2 . 5 2 1 . 5 1 0 . 7 2 . 0 1 8 . 8 0 . 0 — 0 . 0 3 : Morphometric TOTAL LENGTH (TL) MEAN SD CV 6 8 . 8 1 4 . 1 2 0 . 5 6 4 . 7 1 3 . 4 2 0 . 7 6 1 . 3 1 2 . 2 2 0 . 0 5 8 . 9 1 2 . 3 2 0 . 9 5 6 . 0 1 1 . 8 2 1 . 0 4 7 . 4 9 . 8 2 0 . 8 4 4 . 1 9 . 3 2 1 . 2 4 1 . 7 9 . 1 "21.8 3 9 . 3 8 .4 2 1 . 4 3 6 . 5 7 .4 2 0 . 2 3 4 . 0 6 . 6 1 9 . 3 3 1 . 8 6 . 3 1 9 . 9 2 5 . 1 5 . 1 20 .3 and Index Values ARM RATIO (AR) MEAN SD CV 1.12 .03 2 . 7 1.11 .04 3 . 7 1.12 .05 4 . 3 1.13 .03 3 . 1 1.15 .05 4 . 4 2 . 1 9 .07 3 . 4 2 .17 .11 5 . 2 2 .18 .09 4 . 3 2 . 1 9 .10 4 . 7 2 . 1 9 .11 4 . 9 2 .17 .11 4 . 9 8 .00 8 .00 of I n t e r i o r Dougl CENTROMERE INDEX (CI) MEAN SD CV 4 7 . 2 .7 1.5 4 7 . 3 . 9 2 . 0 4 7 . 2 1.1 2 . 4 4 7 . 0 . 7 1.6 4 6 . 6 1.1 2 . 3 3 1 . 4 .7 2 . 3 3 1 . 6 1.1 3 . 5 3 1 . 4 . 9 3 . 0 3 1 . 4 1 .0 3 .2 3 1 . 4 1.1 3 .6 3 1 . 5 1 .0 3 .3 0 . 0 0 . 0 — i - f i r MORPHOLOGICAL INDEX (MI) MEAN SD CV 6 1 . 6 1 3 . 1 2 1 . 3 5 8 . 4 1 3 . 4 2 2 . 9 5 4 . 9 1 2 . 0 2 1 . 8 5 2 . 3 1 1 . 8 2 2 . 5 4 9 . 0 1 1 . 0 2 2 . 4 2 1 . 6 4 . 2 1 9 . 2 2 0 . 4 4 . 4 2 1 . 4 1 9 . 2 4 . 3 2 2 . 4 1 8 . 0 3 . 8 2 1 . 0 1 6 . 8 3 .7 2 2 . 3 1 5 . 6 2 . 9 1 8 . 8 0 . 0 0 . 0 RELATIVE LENGTH (RL) MEAN SD CV 147 3 . 1 2 . 1 138 3 . 1 2 . 3 131 3 . 5 2 .7 125 3 . 1 2 .5 119 3 . 0 2 . 5 101 2 . 1 2 . 1 94 3 . 0 3 .2 89 2 . 8 3 .1 84 2 . 8 3 .3 78 2 . 9 3 .7 73 3 . 0 4 . 1 68 2 . 9 4 . 2 54 2 . 1 3 . 9 25 by a d i f f e r e n c e of 14 u n i t s between t h e t e l o c e n t r i c chromosomes, 12 and 13. T h e r e a r e 11 u n i t s between chromosomes 1 and 2, and 9 u n i t s between chromsomes 2 and 3. The r e m a i n i n g chromosome p a i r s a r e s e p a r a t e d by l e s s t h a n 6 u n i t s . An example o f a chromosome s p r e a d from a t e r m i n a l bud metaphase c e l l f o r I n t e r i o r D o u g l a s - f i r , i s p r e s e n t e d i n F i g u r e 3A. A k a r y o t y p e i s p r e s e n t e d i n F i g u r e 3B. 4.1.2. P_;_ m a c r o c a r p a PM i s c h a r a c t e r i z e d w i t h a h a p l o i d c o m p l i m e n t o f n=12, c o n s i s t i n g o f s i x m e t a c e n t r i c and s i x s u b m e t a c e n t r i c chromosomes s i m i l a r t o t h e A s i a t i c s p e c i e s . A summary of t h e m o r p h o l o g i c a l d a t a and i n d e x v a l u e s o f t h e s p e c i e s a r e f o u n d i n T a b l e 4. D e c r e a s i n g t r e n d s i n l e n g t h a r e f o u n d f o r chromosomes 1-6 and 7-12 (Q) and 1 - 1 2 ( P , TL and M I ) . The AR and CI f o r t h e s i x m e t a c e n t r i c s a r e s i m i l a r , c o n s i s t i n g o f v a l u e s o f 1.07 t o 1.09 and 47.9 t o 48.3 f o r t h e r e s p e c t i v e c h a r a c t e r i s t i c s . Chromosome 11 has a s l i g h t l y s m a l l e r AR v a l u e (2.06) compared t o t h e o t h e r s u b m e t a c e n t r i c s where AR v a l u e s range from 2.10 t o 2.14. CI r a n g e s from 31.9 - 32.3. The maximum d i f f e r e n c e i n RL between n e i g h b o u r i n g chromosomes was f o u n d between chromosomes 6 and 7 (12 u n i t s ) . T h i s was f o l l o w e d by a d i f f e r e n c e of 8 u n i t s between chromosomes 1 and 2. The r e m a i n d e r of t h e n e i g h b o u r i n g p a i r s had r e l a t i v e d i f f e r e n c e s o f 3 t o 5 u n i t s . A p h o t o m i c r o g r a p h of a metaphase t e r m i n a l bud c e l l from PM i s p r e s e n t e d i n F i g u r e 4A. A k a r y o t y p e o f t h e s p e c i e s i s p r e s e n t e d i n F i g u r e 4B. FIGURE 3: A) Metaphase chromosomes (2n=26) from a t e r m i n a l bud c e l l o f I n t e r i o r D o u g l a s - f i r , (2000x), B) K a r y o t y p e o f t h e t h i r t e e n p a i r s o f IDf chromosomes (2000x). CHROMOSOME NUMBER 1 2 3 4 5 6 7 8 9 10 11 12 LONG ARM (Q) MEAN SD CV 37.2 6.4 17.2 35.2 6.2 17.5 33.7 6.0 17.8 32.6 5.8 17.8 31.6 5.5 17.4 30.6 5.5 18.1 35.6 5.6 16.8 33.6 5.7 17.0 31.8 5.3 16.7 29.9 4.7 15.8 27.9 4.7 16.9 26.5 4.6 17.3 TABLE 4: SHORT ARM (P) MEAN SD CV 34.8 6.1 17.4 32.7 5.6 17.2 31.3 5.5 17.5 30.3 5.4 17.6 29.2 4.9 16.7 28.0 4.8 16.9 16.9 3.0 17.5 15.8 2.7 17.2 15.0 2.5 16.8 14.0 2.3 16.2 13.5 2.1 15.9 12.6 2.1 16.6 M o r p h o m e t r i c and TOTAL LENGTH (TL) MEAN SO CV 72.0 12.4 17.2 67.8 11.7 17.3 65.1 11.4 17.6 62.9 11.1 17.6 60.8 10.3 17.0 58.6 10.2 17.5 52.4 8.8 16.9 49.4 8.4 16.9 46.8 7.8 16.6 44.0 6.9 15.7 41.3 6.8 16.6 39.1 6.6 17.0 Index V a l u e s o f P. ARM RATIO (AR) MEAN SD CV 1.07 .04 3.3 1.08 .03 3.2 1.08 .03 3.2 1.07 .04 3.8 1.08 .04 3.4 1.09 .05 4.2 2.11 .12 5.7 2.13 .10 4.5 2.12 .09 4.3 2.14 .10 4.5 2.06 .09 4.5 2.10 .09 4.1 m a c r o c a r p a . CENTROMERE INDEX (CI) MEAN SD CV 48.3 .8 1.7 48.2 .8 1.7 48.2 .8 1.7 48.2 1.0 2.0 48.1 .8 1.8 47.9 1.1 2.2 32.3 1.2 3.7 32.0 1.0 3.0 32.1 .9 2.9 31.9 1.0 3.0 32.7 1.0 3.1 32.3 .9 2.8 MORPHOLOGICAL INDEX (MI) MEAN SD CV 67.2 11.9 17.7 63.0 10.9 17.3 60.4 10.6 17.5 58.6 10.5 17.9 56.2 9.3 16.6 53.7 9.0 16.7 24.9 4.5 18.0 23.2 4.1 17.5 22.1 3.8 17.1 20.6 3.5 17.0 20.0 3.1 15.7 18.6 3.1 16.0 RELATIVE LENGTH (RL) MEAN SD CV 131 3.4 2.6 123 3.1 2.5 118 3.1 2.6 114 2.6 2.3 110 2.1 1.9 107 1.9 1.8 95 1.9 2.0 90 2.5 2.7 85 3.5 4.1 80 3.5 4.3 75 2.8 3.7 71 2.3 3.3 A) Metaphase chromosomes (2n=24) from a terminal bud c e l l of P_:_ macrocarpa, (2000x) , B) Karyotype of the twelve pairs of PM chromosomes, (2000x). 29 4.1.3. f o r r e s t i i PF c o n t a i n s a h a p l o i d c o m p l i m e n t of n=12, c o n s i s t i n g of s i x m e t a c e n t r i c and s i x s u b m e t a c e n t r i c chromosomes. G e n e r a l l y Q d i s p l a y s a d e c r e a s i n g t r e n d f o r chromosomes 1-6 and 7-12. P and TL v a l u e s d e c r e a s e f o r chromosomes 1-12 ( T a b l e 5 ) . The e x c e p t i o n s a r e t h e Q v a l u e s o f chromosomes 4 (Q=32.0) and 5 (Q=32.3) and t h e P v a l u e s of chromosomes 2 (P=30.4) and 3 (P=31.4), where t h e t r e n d i s r e v e r s e d . Of chromosome 3 (P=31.4). One of t h e d i s t i n g u i s h i n g f e a t u r e s of t h i s s p e c i e s i s t h e c e n t r o m e r e p o s i t i o n of chromosome 2. The chromosome has an AR o f 1.25 compared t o t h e AR of 1.10, 1.09, 1.02, 1.12, and 1.03 o f th e o t h e r m e t a c e n t r i c s i n t h i s s p e c i e s . The u n e q u a l arm l e n g t h s of t h i s chromosome make i t r e l a t i v e l y e a s y t o d i s t i n g u i s h from t h e o t h e r m e t a c e n t r i c s . CI has a low e r v a l u e f o r chromosome 2 (44.4) compared t o t h e o t h e r m e t a c e n t r i c s w h i c h have CI v a l u e s r a n g i n g from 47.1 t o 49.5. MI e x h i b i t s a d e c r e a s i n g t r e n d f o r t h e s u b m e t a c e n t r i c s (chromosomes 7-12) but not t h e m e t a c e n t r i c chromosomes. Chromosome 1 has an MI of 67.9, w h i l e chromosomes 2-6 have v a l u e s of 54.7, 60.2, 62.0, 54.1 and 55.9, f o l l o w i n g no a p p a r e n t t r e n d . From t h e RL i t can be n o t e d t h a t t h e maximum d i f f e r e n c e between n e i g h b o u r i n g p a i r s o c c u r s between t h e s h o r t e s t m e t a c e n t r i c , chromosome 6 and t h e l o n g e s t s u b m e t a c e n t r i c , chromosome 7 (18 u n i t s ) . Among . t h e m e t a c e n t r i c s maximum d i f f e r e n c e i n l e n g t h o c c u r s between chromosomes 1 and 2 (10 u n i t s ) and t h e minimum d i f f e r e n c e between 3 and 4 (4 u n i t s ) . TABLE 5: Morphometric and Index Values of P;_ for rest i i . CHROMOSOME NUMBER LONG ARM (Q) MEAN SD CV SHORT ARM (P) MEAN SD CV TOTAL LENGTH (TL) MEAN SD CV ARM RATIO (AR) MEAN SD CV CENTROMERE INDEX (CI) MEAN SD CV MORPHOLOGICAL INDEX (MI) MEAN SD CV RELATIVE LENGTH (RL) MEAN SD CV 38.9 6.0 15.3 35.5 5.8 16.3 74.4 11.7 15.8 1.10 .03 2.4 47.7 .59 1.2 67.9 11.9 16.9 139 1.6 1.1 38.2 6.2 16.2 30.4 7.6 19.4 68.6 10.8 15.8 1.25 .07 5.6 44.4 1.40 3.2 54.7 8.9 16.3 129 1.8 1.4 34.2 5.3 15.3 31.4 5.1 16.3 65.5 10.3 15.6 1.09 .04 3.6 47.8 .88 1.8 60.2 10.3 17.2 123 1.8 1.5 32.0 4.9 15.4 31.3 5.0 16.1 63.3 9.9 15.7 1.02 .02 2.3 49.5 .56 1.1 62.0 10.3 16.0 119 1.9 1.6 32.3 5.2 16.1 28.7 4.4 15.5 60.9 9.6 15.7 1.12 .04 3.8 47.1 .96 2.1 54.1 8.3 15.4 114 1.7 1.5 29.3 4.6 15.8 28.4 4.3 15.0 57.6 8.8 15.3 1.03 .03 2.8 49.2 .71 1.4 55.9 8.3 14.9 108 1.8 1.7 34.3 5.7 16.7 14.1 2.3 16.2 48.3 7.8 16.1 2.44 .19 7.6 29.1 1.57 5.4 19.8 3.3 16.8 90 1.5 1.6 31.8 4.7 14.8 12.9 2.5 19.1 44.7 7.0 15.6 2.49 .19 7.5 28.8 1.66 5.8 18.2 3.9 21.3 84 2.1 2.5 29.9 4.8 15.9 12.7 2.3 17.8 42.6 6.8 15.8 2.38 .19 7.9 29.7 1.65 5.4 18.3 3.4 19.2 80 1.4 1.7 10 29.0 4.6 15.9 12.0 2.2 18.1 40.9 9.0 20.0 2.44 .19 7.7 29.2 1.60 5.5 16.9 3.3 19.4 77 1.4 1.8 11 27.0 4.5 15.6 11.5 1.8 15.6 38.5 6.2 16.1 2.35 .11 4.7 29.9 .99 3.3 16.4 2.5 15.5 72 1.0 1.4 12 24.3 4.1 16.7 10.8 1.7 15.4 35.1 5.6 16.0 2.26 .15 6.8 30.7 1.42 4.7 15.5 2.4 15.4 66 2.0 3.0 Co O 31 In t h e s u b m e t a c e n t r i c s maximum d i f f e r e n c e o c c u r s between chromosomes 7 and 8, and 11 and 12 (6 u n i t s e ach) and t h e l e a s t between 9 and 10 (3 u n i t s ) . A p h o t o m i c r o g r a p h o f a t e r m i n a l bud metaphase c e l l and a k a r y o t y p e of t h e PF s p e c i e s i s p r e s e n t e d i n F i g u r e 5. The s h o r t arm o f chromosome 2 ( F i g u r e 5B) i s v i s i b l y s m a l l e r t h a n t h e l o n g arm of t h i s chromosome. 4.1.4. Pj_ s i n e n s i s PS has a h a p l o i d c o m p l i m e n t o f n=12 i n c l u d i n g s i x m e t a c e n t r i c s and s i x s u b m e t a c e n t r i c s . A d e c r e a s i n g t r e n d i s o b s e r v e d i n chromosomes 1 t h r o u g h 12 f o r P, TL and MI. Chromosomes 1 t o 6 and 7 t o 12 e x p r e s s a d e c r e a s i n g t r e n d f o r Q ( T a b l e 6 ) . F o r t h e m e t a c e n t r i c chromosomes, AR r a n g e s from. 1.05 t o r. 07 and CI from 48.3 t o 48.9. The AR and CI v a l u e s a r e a l s o q u i t e u n i f o r m f o r t h e s u b m e t a c e n t r i c s , where AR r a n g e s from 2.09 t o 2.16 and CI from 31.7 t o 32.4. Maximum d i f f e r e n c e i n RL between n e i g h b o u r i n g chromosomes o c c u r s between chromosomes 6 and 7 (18 u n i t s ) . The maximum d i f f e r e n c e i n RL w i t h i n a m o r p h o l o g i c a l c l a s s o c c u r s between chromosomes 1 and 2 (9 u n i t s ) i n t h e m e t a c e n t r i c s and 7 and 8 (9 u n i t s ) i n t h e s u b m e t a c e n t r i c s . The r e l a t i v e d i f f e r e n c e between t h e r e m a i n i n g chromosome p a i r s i s 4 t o 6 u n i t s . A PS metaphase r o o t t i p c e l l i s p r e s e n t e d i n F i g u r e 6A. A k a r y o t y p e c o m p a r i n g t h e 12 chromosome p a i r s i s l o c a t e d i n F i g u r e 6B. 32 « J7 \) O 17 » B 7 8 9 10 11 12 FIGURE 5: A) Metaphase chromosomes (2n=24) from a t e r m i n a l bud c e l l o f P. f o r r e s t i i , (2000x), B) K a r y o t y p e o f t h e t w e l v e p a i r s o f PF chromosomes, (2000x). TABLE 6 : M o r p h o m e t r i c and Index V a l u e s o f s i n e n s i s . CHROMOSOME NUMBER LONG ARM (fi) MEAN SD C V SHORT ARM (P) MEAN SD CV TOTAL LENGTH (TL) MEAN SD CV ARM RATIO (AR) MEAN SD CV CENTROMERE INDEX (CI) MEAN SD CV MORPHOLOGICAL INDEX (MI) MEAN SD CV RELATIVE LENGTH (RL) MEAN SD CV 4 4 . 4 8 .9 20 .1 4 2 . 0 8 .3 20 .0 86 .4 1 7 . 2 1 9 . 9 1.06 .05 5 .1 4 8 . 7 .57 1 .2 8 1 . 9 1 6 . 6 2 0 . 2 140 4 . 3 3 . 1 4 1 . 7 7 .8 1 8 . 8 3 9 . 2 7 . 6 19 .4 8 0 . 9 1 5 . 4 1 9 . 0 1.07 .04 3 .8 4 8 . 4 .94 1.9 7 6 . 0 1 5 . 2 2 0 . 6 1 3 1 . . 2 . 4 1.9 3 9 . 8 7 . 5 1 8 . 8 3 7 . 1 7 . 0 18 .7 7 6 . 9 1 4 . 3 1 8 . 6 1.07 .03 5 .1 4 8 . 3 .98 2 . 5 7 1 . 8 1 3 . 6 1 9 . 0 125 2 . 3 1.8 3 7 . 8 6 . 9 18 .4 3 6 . 0 6 . 4 1 7 . 9 7 3 . 7 1 3 . 3 1 8 . 1 1.05 .04 3 .7 4 8 . 9 .79 1.6 7 0 . 3 1 2 . 5 1 7 . 8 120 2 . 1 1.7 3 6 . 5 7 . 1 19 .4 3 4 . 3 6 . 5 1 9 . 1 70 .7 1 3 . 5 1 9 . 1 1.07 .04 3 .7 4 8 . 4 .88 1.8 6 6 . 5 1 2 . 5 1 9 . 3 115 2 . 5 2 . 2 3 4 . 7 7 . 0 2 0 . 0 3 2 . 6 6 . 4 19 .7 6 7 . 4 1 3 . 3 1 9 . 5 1.07 .04 3 .7 4 8 . 4 . 91 1 .9 6 3 . 3 1 2 . 5 1 9 . 8 109 3 . 1 2 . 8 3 8 . 2 7 . 0 18 .3 1 7 . 7 3 .3 1 8 . 5 5 5 . 9 1 0 . 2 1 8 . 2 2 . 1 6 . .11 5.3 3 1 . 7 1.13 3 . 6 2 6 . 0 4 . 9 1 8 . 8 91 2 . 2 2 . 4 34 .4 6 . 4 1 8 . 6 1 6 . 4 3 . 0 18 .3 5 0 . 8 9 .3 1 8 . 3 2 . 0 9 .11 5 .3 3 2 . 4 1 .19 3 .7 2 4 . 3 4 . 5 1 8 . 6 82 2 . 5 3 . 0 3 2 . 7 6 . 2 1 9 . 1 1 5 . 3 3 .4 22 .2 4 8 . 0 9 .5 1 9 . 8 2 . 1 5 .15 6 .8 3 1 . 8 1 .49 4 . 7 2 2 . 5 5 .3 2 3 . 7 78 2 . 1 2 . 4 10 3 1 . 0 6 .4 2 0 . 5 1 4 . 5 2 . 7 18 .8 4 5 . 5 9 . 0 2 0 . 0 2 . 1 5 .14 6 .3 3 1 . 9 1.37 4 . 3 2 1 . 2 4 . 0 1 8 . 8 74 2 . 5 3 . 5 11 2 9 . 1 5 .7 19 .7 1 3 . 9 2 . 6 1 8 . 9 4 2 . 9 8 .2 1 9 . 9 2 . 1 0 .13 6 .3 3 2 . 4 1 .40 4 . 3 2 0 . 5 3 . 9 1 9 . 0 70 2 . 1 3 . 0 12 2 6 . 7 5 .1 1 8 . 5 1 2 . 5 2 . 2 17 .8 3 9 . 2 7 .2 1 8 . 4 2 .14 .15 7 . 1 3 1 . 9 1.51 4 . 7 1 8 . 3 3 .3 1 8 . 3 64 2 . 4 3 .7 Co OJ 34 FIGURE 6: A) Metaphase chromosomes (2n=24) from a root tip cell of P^ sinensis, (2000x) B) Karyotype of the twelve pairs of PS chromosomes (2000x). 35 4.1.5. g a u s s e n i i T h i s was t h e f i r s t r e p o r t e d k a r y o t y p e a n a l y s i s o f PG. T h i s s p e c i e s f o l l o w e d t h e t r e n d s e t by t h e o t h e r A s i a t i c s p e c i e s by c o n t a i n i n g a h a p l o i d c o m p l i m e n t of n=12 c o n s i s t i n g of s i x m e t a c e n t r i c s and s i x s u b m e t a c e n t r i c s . The v a l u e s f o r t h e d i f f e r e n t chromosome p a r a m e t e r s measured a r e summarized i n T a b l e 7, A d e c r e a s i n g t r e n d i s o b s e r v e d i n Q f o r chromosomes 1-6 and 7-12 and i n P, TL and MI f o r chromosomes 1-12. AR and CI a r e c o n s i s t e n t f o r t h e m e t a c e n t r i c s and s u b m e t a c e n t r i c s , w i t h v a l u e s o f 1.03 t o 1.05 f o r AR and 48.8 t o 49.2 f o r CI- i n t h e m e t a c e n t r i c s and AR o f 2.27 t o 2.32 and CI of 30.2 t o 30.6 f o r t h e s u b m e t a c e n t r i c s . The maximum d i f f e r e n c e i n t h e mean RL between two n e i g h b o u r i n g chromosome p a i r s i s 18 u n i t s between chromosome 6 ( m e t a c e n t r i c ) and chromosome 7 ( s u b m e t a c e n t r i c ) . The maximum d i f f e r e n c e i n RL between n e i g h b o u r i n g m e t a c e n t r i c s i s between chromosomes 1 and 2 (10 u n i t s ) and between 7 and 8 (7 u n i t s ) i n th e s u b m e t a c e n t r i c s . The d i f f e r e n c e i n RL between t h e r e m a i n i n g chromosome p a i r s i s q u i t e c o n s t a n t a t 4 t o 6 u n i t s . A p h o t o m i c r o g r a p h o f a chromosome s p r e a d i n a PG r o o t t i p c e l l a t metaphase i s l o c a t e d i n F i g u r e 7A. A k a r y o t y p e of t h e s p e c i e s i s l o c a t e d i n F i g u r e 7B. TABLE 7: Morphometric and Index V a l u e s o f P. g a u s s e n i i . CHROMOSOME NUMBER . 4 10 11 12 LONG ARM (Q) MEAN SD CV SHORT ARM (P) MEAN SD CV TOTAL LENGTH (TL) MEAN SD CV ARM RATIO (AR) MEAN SD CV CENTROMERE INDEX (CI) MEAN SD CV MORPHOLOGICAL INDEX (MI) MEAN SD CV 43.2 8.8 20.8 41.2 8.5 20.6 84.4 17.4 20.7 1.05 .02 2.3 48.8 .57 1.2 80.4 16.6 20.6 40.1 8.3 20.7 38.6 8.4 21.7 78.7 16.6 21.2 1.04 .03 3.0 49.0 .74 1.5 75.6 16.9 22.3 38.3 8.0 20.5 36.6 7.3 20.0 74.9 15.1 20.1 1.05 .04 3.6 48.9 .88 1.8 71.6 14.3 19.9 36.7 7.0 19.0 35.3 4.0 11.0 72.0 14.1 19.5 1.04 .04 4.1 48.9 .99 2.0 69.2 14.5 21.0 35.3 6.9 19.4 33.8 7.2 21.3 69.1 14.0 20.3 1.05 .04 3.6 48.8 .88 1.8 66.0 14.7 22.3 33.0 6.8 20.7 31.9 6.5 20.3 65.0 13.3 20.4 1.03 .03 3.0 49.2 .74 1.5 63.0 12.7 20.2 37.5 7.7 20.5 16.4 3.6 22.0 53.9 11.2 20.8 2.29 .12 5.4 30.4 1.11 3.7 23.6 5.4 22.7 34.4 7.2 20.9 15.2 3.2 21.0 49.5 10.3 20.8 2.27 .09 4.1 30.6 .87 2.8 21.8 4.6 21.7 32.6 6.7 20.6 14.4 3.1 21.7 47.0 9.8 20.9 2.27 .10 4.4 30.6 .91 3.0 20.8 4.6 22.2 30.9 6.5 20.9 13.6 3.0 21.8 44.5 9.4 20.9 2.28 .10 4.4 30.5 .91 2.9 19.6 4.2 22.1 29.4 6.5 22.1 12.7 2.6 20.5 42.0 9.0 21.5 2.32 .11 4.9 30.2 1.00 3.3 18.1 3.7 20.4 26.7 2.4 20.2 11.7 2.4 20.2 38.8 7.7 19.9 2.28 .11 5.0 30.5 1.05 3.4 16.8 3.5 20.5 RELATIVE LENGTH (RL) MEAN SD CV 141 2.6 1.8 131 2.2 1.7 125 1.8 1.5 120 2.8 2.3 115 2.1 1.8 108 2.1 1.9 90 2.6 2.9 83 2.4 2.9 78 1.9 2.4 74 1.9 2.6 70 2.4 3.5 64 1.7 2.6 Lo CM 37 38 4.1.6. j a p o n i c a P J , a n a t i v e o f J a p a n has a k a r y o t y p e s i m i l a r t o t h a t of t h e C h i n e s e s p e c i e s , w i t h a h a p l o i d s e t o f n=12 c o n s i s t i n g of s i x m e t a c e n t r i c and s i x s u b m e t a c e n t r i c p a i r s of chromosomes. The summary of m o r p h o m e t r i c and i n d e x v a l u e s i s f o u n d i n T a b l e 8. A d e c r e a s i n g t r e n d i n Q i s f o u n d f o r chromosomes 1-6 and 7-12 and chromosomes 1-12 f o r P, TL and MI. The AR and CI v a l u e s o f t h e m e t a c e n t r i c chromosomes a r e q u i t e u n i f o r m r a n g i n g f r o m 1.04 t o 1.07 and 48.4 t o 49.0 f o r AR and CI r e s p e c t i v e l y . A s i m i l a r h o m o g e n e i t y was o b s e r v e d f o r t h e s u b m e t a c e n t r i c s , where AR v a l u e s r a n g e between 2.30 and 2.36 and CI between 29.& and 30.3. The maximum d i f f e r e n c e i n RL o c c u r s between chromosomes 6 and 7 (17 u n i t s ) . The r e l a t i v e d i f f e r e n c e s between t h e o t h e r n e i g h b o u r i n g chromosomes a r e s i m i l a r , w i t h t h e maximum d i f f e r e n c e o c c u r r i n g between chromosomes 1 and 2 (8 u n i t s ) and chromosomes 7 and 8 (7 u n i t s ) and t h e minimum d i f f e r e n c e between chromosomes 4 and 5, and 9 and 10 (4 u n i t s e a c h ) . A metaphase r o o t t i p c e l l o f P J i s f o u n d i n F i g u r e 8A. A k a r y o t y p e of t h e s p e c i e s i s p r e s e n t e d i n F i g u r e 8B. 4.1.7. P^ _ w i l s o n i a n a PW w i t h a h a p l o i d s e t o f n=12, i n c l u d i n g s i x m e t a c e n t r i c s and s i x s u b m e t a c e n t r i c s does not d e v i a t e f r o m t h e k a r y o t y p e common t o t h e o t h e r A s i a t i c s p e c i e s . The m o r p h o m e t r i c and i n d e x v a l u e s a r e summarized i n T a b l e 9. CHROMOSOME NUMBER 1 2 3 4 5 6 7 a 9 10 n 12 LONG ARM (Q) MEAN SD CV 43.4 4.7 10.9 40.9 4.5 11.7 38.9 4.4 11.4 37.1 4.2 11.3 35.7 3.8 10.6 34.3 4.7 13.7 38.6 5.0 12.9 35.9 4.6 12.8 33.9 4.2 12.4 32.0 3.8 11.8 30.0 3.5 11.7 27.3 3.6 13.0 TABLE SHORT ARM' (P) MEAN SD CV 41.0 4.5 11.0 38.4 4.5 11.7 36.9 4.0 10.9 35.7 4.0 11.0 34.1 3.8 11.1 32.9 7.3 22.1 16.7 2.2 12.9 15.5 1.8 11.7 14.4 1.7 11.5 13.6 1.7 12.1 13.0 1.5 11.6 11.9 1.5 12.4 8: Morphometric s TOTAL LENGTH (TL) MEAN SD CV 84.3 9.1 9.1 79.3 8.9 11.2 75.8 8.4 11.1 72.8 8.1 11.1 69.8 7.5 10.7 67.2 10.3 15.3 55.3 7.0 12.6 51.5 6.4 12.3 48.3 5.8 12.0 45.6 5.3 11.6 43.0 4.9 11.4 39.2 4.9 12.6 Index Values of ARM RATIO (AR) MEAN SD CV 1.06 .05 4.6 1.07 .04 3.5 1.05 .03 2.7 1.04 .03 2.6 1.05 .04 3.7 1.05 .04 3.8 2.32 .15 6.6 2.31 .10 4.2 2.36 .13 5.6 2.36 .11 4.6 2.32 .10 4.4 2.30 .11 4.7 P. japonica. CENTROMERE INDEX (CI) MEAN SD CV 48.6 1.1 2.2 48.4 .9 1.8 48.7 .7 1.4 49.0 .7 1.3 48.8 .9 1.9 48.8 .9 1.8 30.2 1.4 4.5 30.2 .9 2.9 29.8 1.2 4.0 29.8 1.0 3.2 30.1 .9 3.1 30.3 1.0 3.3 MORPHOLOGICAL INDEX (MI) MEAN SD CV 79.7 9.2 11.5 74.4 9.2 12.4 71.9 7.8 10.9 69.9 7.8 11.2 66.6 7.8 11.7 65.7 7.8 11.6 23.9 3.2 13.5 22.2 2.6 11.6 20.5 2.4 11.8 19.3 2.4 12.7 18.5 2.2 11.9 17.0 2.1 12.6 RELATIVE LENGTH (RL) MEAN SD CV 138 3.5 2.5 130 2.3 1.8 124 2.4 2.0 119 2.6 2.2 115 3.1 2.7 108 2.4 2.3 91 2.9 3.2 84 2.9 3.4 79 2.6 3.3 75 2.8 3.7 70 2.8 4.0 64 2.3 3.7 40 1 2 3 4 5 6 j ( {1 (I ( ( » <? 7 8 9 10 11 12 FIGURE 8: A) Metaphase chromosomes (2n=24) from a r o o t t i p c e l l o f I\_ j a p o n i c a , (2000x) , B) K a r y o t y p e o f t h e t w e l v e p a i r s o f P J chromosomes (2000x). CHROMOSOME NUMBER 1 2 3 4 S 6 7 B 9 10 11 12 LONG ARM (Q) MEAN SO CV 38.8 7.8 20.1 36.9 7.5 20.3 35.3 6.5 18.5 34.2 6.2 18.2 33.0 5.6 17.1 31.5 5.5 17.6 34.1 6.4 18.7 31.7 5.6 17.6 30.2 5.2 17.4 28.8 5.3 18.5 27.2 4.8 17.6 24.5 4.3 17.5 TABLE SHORT ARM (P) MEAN. SD CV 35.9 6.5 18.0 33.6 6.0 17.9 32.6 5.8 17.9 31.5 5.6 17.7 30.2 5.2 17.2 28.9 4.6 16.0 14.5 2.6 18.0 13.4 2.3 16.9 12.6 2.1 16.5 12.1 1.8 14.6 11.4 1.7 15.1 10.7 1.7 16.1 9: Morphometric TOTAL LENGTH (TL) MEAN SD CV 74.6 6.5 18.0 70.5 13.5 19.0 67.8 12.3 18.1 65.7 11.6 17.7 63.1 10.7 17.0 60.5 10.1 16.6 48.7 8.8 18.1 45.0 7.7 17.1 42.8 7.2 16.9 40.9 7.0 17.1 38.6 6.4 16.6 35.2 5.9 16.9 and Index V a l u e s i ARM RATIO (AR) MEAN SD CV 1.08 .04 3.7 1.10 .05 4.9 1.08 .04 3.9 1.09 .06 5.9 1.09 .05 4.8 1.09 .06 5.3 2.35 .17 7.4 2.37 .14 6.0 2.40 .15 6.1 2.37 .15 6.5 2.39 .17 6.9 2.29 .17 7.2 P. w i l s o n i a n a . CENTROMERE INDEX (CI) MEAN SO CV 48.1 .9 1.9 47.7 1.2 2.6 48.0 1.0 2.0 47.9 1.4 3.0 47.8 1.2 2.5 47.9 1.3 2.7 29.9 1.5 5.3 29.7 1.2 4.2 29.5 1.3 4.2 29.7 1.4 8.1 29.6 1.5 5.2 30.5 1.6 5.2 MORPHOLOGICAL INDEX (MI) MEAN SD CV 69.1 11.9 17.2 64.1 11.0 17.2 62.6 11.2 18.0 60.5 11.0 18.2 57.8 10.0 17.7 55.3 8.8 15.8 20.7 3.8 18.6 19.7 3.2 17.0 17.8 3.0 16.8 17.2 2.4 13.8 16.2 2.4 14.9 15.4 2.5 16.2 RELATIVE LENGTH (RL) MEAN SD CV 137 3.0 2.6 129 3.1 2.4 125 2.2 1.8 121 2.2 1.8 116 2.1 1.8 111 2.5 2.3 89 2.7 3.0 83 2.2 2.6 79 2.3 2.9 75 1.9 2.5 71 2.1 3.0 65 2.4 3.6 42 A d e c r e a s i n g t r e n d i s o b s e r v e d i n chromosomes 1-12 f o r P, TL and MI and i n chromosomes 1-6 and 7-12 f o r Q. The AR of t h e m e t a c e n t r i c s r a n g e s from 1.08 t o 1.10 and t h e s u b m e t a c e n t r i c s from 2.29 t o 2.40. The CI f o r t h e m e t a c e n t r i c s (47.7 - 48.1) and s u b m e t a c e n t r i c s (29.5 - 30.5) a r e a l s o q u i t e s i m i l a r . T h e r e i s a l a r g e d i f f e r e n c e i n RL between chromosomes 6 and 7 (22 u n i t s ) . The maximum r e l a t i v e d i f f e r e n c e between n e i g h b o u r i n g m e t a c e n t r i c s i s 8 u n i t s between chromosomes 1 and 2. The d i f f e r e n c e between t h e r e m a i n i n g chromosome p a i r s v a r y between 4 and 6 u n i t s . A m i c r o p h o t o g r a p h o f a chromosome s p r e a d o f PW metaphase r o o t t i p - c e l l i s f o u n d i n F i g u r e 9A. A k a r y o t y p e of t h e s p e c i e s i s l o c a t e d i n F i g u r e 9B. 4.1 .8 . S e c o n d a r y C o n s t r i c t i o n s Many " s e c o n d a r y c o n s t r i c t i o n - l i k e " o b j e c t s were o b s e r v e d i n t h e d i f f e r e n t s p e c i e s . To d i f f e r e n t i a t e SC from a r t i f a c t s o n l y t h o s e o b s e r v e d i n g r e a t e r t h a n o r e q u a l t o 70% o f t h e c e l l s w i l l be d i s c u s s e d . E a c h s p e c i e s c o n t a i n e d a t l e a s t one chromosome w i t h a c o n s i s t e n t SC. F o r e a c h s p e c i e s , t h e p o s i t i o n and f r e q u e n c y o f t h e SC a r e t a b u l a t e d i n T a b l e 10. X X K I i i> 1 2 3 4 5 6 I) (J il I) » U 7 8 9 10 11 12 FIGURE 9: A) Me taphase chromosomes (2n=24) f rom a r o o t t i p c e l l o f I\_ w i l s o n i a n a , (2000x) , B) K a r y o t y p e o f t h e t w e l v e p a i r s o f PW chromosome's (2000x) . 44 TABLE 10: The L o c a t i o n and F r e q u e n c y of S e c o n d a r y C o n s t r i c t i o n s SPECIES LOCATION* FREQUENCY (%) CDf 3 L 56 83 10 L 24 70 IDf 3 L 56 73 10 L 23 73 PM 4 L 60 73 2 L 57 70 PF 4 L 51 95 6 L 49 75 P J 4 L 53 98 2 L 56 70 PS 4 L 48 88 PG * - 4 L 48 95 PW 4 L 54 95 * See M a t e r i a l And Methods f o r e x p l a n a t i o n o f L o c a t i o n . Two SC were o b s e r v e d i n CDf, one on t h e l o n g arm o f chromosome 3 and t h e o t h e r on t h e l o n g arm of chromosome 10, o c c u r r i n g a t f r e q u e n c i e s of 83 and 70%, r e s p e c t i v e l y . Two SC were c o n s i s t e n t l y f o u n d i n I D f , b o t h o c c u r r i n g a t f r e q u e n c i e s of 73% on t h e l o n g arms of chromosomes 3 and 10. The p o s i t i o n of t h e SC on t h e chromosomes were v e r y s i m i l a r , i f n o t i d e n t i c a l f o r t h e two v a r i e t i e s , w i t h t h e SC on chromosome 3 o c c u r r i n g a t p o s i t i o n 56 (56% of t h e d i s t a n c e f r o m t h e c e n t r o m e r e t o t h e t e l o m e r e o f t h a t arm) f o r e a c h v a r i e t y and t h e SC on chromosome 10 s i t u a t e d a t p o s i t i o n 24 f o r CDf and 23 f o r IDf ( T a b l e 10). 45 Two SC were d e t e c t e d i n PM, one o c c u r r i n g a t p o s i t i o n 60 o f t h e l o n g arm of chromosome 4 and t h e o t h e r a t p o s i t i o n 57 o f t h e l o n g arm of chromosome 2. The o b s e r v e d f r e q u e n c i e s o f o c c u r r e n c e were 73 and 70%, r e s p e c t i v e l y . Two SC were o b s e r v e d i n P J , s i t u a t e d on t h e l o n g arms o f chromosomes 4 and 2 a t p o s i t i o n 53 and 56, r e s p e c t i v e l y . The f r e q u e n c y o f o c c u r r e n c e was 98% f o r t h e SC on chromosome 4 and 70% f o r t h e SC on chromosome 2. PS, PG and PW c o n t a i n e d one SC e a c h on t h e l o n g arm o f chromosome 4. The c o n s t r i c t i o n s o c c u r r e d a t p o s i t i o n 48 f o r PS and PG and a t p o s i t i o n 54 f o r Psw. The f r e q u e n c i e s o f o c c u r r e n c e were 95% f o r PW and PG and 88% f o r PS. An i d i o g r a m 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 s o f t h e chromosomes and t h e number and p o s i t i o n of t h e SC f o r t h e s p e c i e s and v a r i e t i e s i s p r e s e n t e d i n F i g u r e 10. The most n o t i c e a b l e d i f f e r e n c e s a r e t h e v a r i a t i o n i n chromosome number between D o u g l a s - f i r (n=13) and t h e r e m a i n i n g s p e c i e s (n=12), t h e v a r i a t i o n i n t h e number and p o s t i i o n o f SC and t h e d i v e r g e n c e from t h e d e c r e a s i n g l e n g t h t r e n d f o r t h e m e t a c e n t r i c chromosomes of PF. 4.2. Compari son of 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 (n=13) by  T - t e s t A t - t e s t was employed t o d e t e r m i n e i f t h e r e were any s i g n i f i c a n t l y d i f f e r e n t chromosomal c h a r a c t e r i s t i c s between 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 . The se v e n m o r p h o l o g i c a l c h a r a c t e r s , Q, P, TL, AR, C I , MI-and RL were compared f o r e a c h 46 chromosome and t h e r e s u l t s a r e t a b u l a t e d i n T a b l e 11. The t -v a l u e s a r e e x p r e s s e d as n o n - s i g n i f i c a n t , s i g n i f i c a n t , p<0.05 and h i g h l y s i g n i f i c a n t , p < 0 . 0 l . Q13 ( l o n g arm of chromosome 13) and TL13 a r e non-s i g n i f i c a n t . Q10, P10, TL10, Q11, TL11, Q12 and TL12 a r e s i g n i f i c a n t l y d i f f e r e n t (p<0.05), w h i l e t h e r e m a i n i n g Q, P and TL t - v a l u e s a r e h i g h l y s i g n i f i c a n t ( p <0.0l) between t h e two v a r i e t i e s . The t - v a l u e s f o r a l l AR and CI v a r i a b l e s a r e non-s i g n i f i c a n t . MI5 i s s i g n i f i c a n t l y d i f f e r e n t (p<0.05), w h i l e M11 t o MI4 and -MI 6 t o MI 11 a r e h i g h l y s i g n i f i c a n t ( p < 0 . 0 l ) . MI 12 and M113 a r e n o t i n c l u d e d s i n c e t h e i r v a l u e s a r e b o t h 0.00. RL3 d i f f e r s s i g n i f i c a n t l y a t t h e 5% l e v e l , w h i l e RL1, 6, 10, 11 and 12 a r e h i g h l y s i g n i f i c a n t a t t h e 1% l e v e l . RL2, 4, 5, 7, 8, 9 and 13 a r e not s i g n i f i c a n t l y d i f f e r e n t between CDf and I D f . The h i g h e s t t - v a l u e i s a t t r i b u t e d t o RL6 ( 6 . 5 7 ) , f o l l o w e d by RL13 ( 5 . 6 4 ) , P6 (3.79) and MI 6 ( 3 . 7 7 ) . Based on t h e r e s u l t s of th e t - t e s t i t i s p o s s i b l e t o d i f f e r e n t i a t e between CDf and IDf u s i n g t h e c h a r a c t e r i s t i c s t h a t were s i g n i f i c a n t l y d i f f e r e n t . 47 Coastal Douglas-fir I n t e r i o r Douglas-fir FIGURE 10: Idiograms of the Pseudotsuga species showing the p o s i t i o n of the secondary c o n s t r i c t i o n s . TABLE 11: Comparison of Coasta l and In ter ior D o u g l a s - f i r . VARIABLE CDF IDF T-VALUE VARIABLE CDF IDF T-VALUE VARIABLE Ql 31.40 36.33 3.30** Q4 27.10 31.18 3.15** Q7 PI 27.90 32.50 3.31** P4 23.93 27.70 3.12** P7 TL1 59.30 68.83 3.32** TL4 51.03 58.88 3.15** TL7 AR1 1.13 1.12 .89 AR4 1.14 1.13 .53 AR7 CI1 47.00 47.18 .80 CI4 46.87 46.98 .44 CI7 M i l 52.76 61.60 3.26** MI4 45.10 52.33 3.07** MI7 R L l 145 147 3.41** RL4 124 125 1.47 RL7 Q2 29.26 34.03 3.15** Q5 25.92 29.85 3.11** 08 P2 26.45 30.68 3.20** P5 23.13 26.13 2.58* P8 TL2 56.08 64.70 3.19** TL5 49.05 55.98 2.87** TL8 AR2 1.12 1.11 .68 AR5 1.13 1.15 1.64 AR8 CI2 47.19 47.33 .66 CI5 47.10 46.63 1.72 CI8 MI2 50.11 58.37 3.17** MI5 43.80 49.03 2.31* MI8 RL2 137 138 1.74 RL5 120 119 .48 RL8 Q3 27.95 32.35 3.38** Q6 27.48 32.55 3.64** 09 P3 25.03 28.95 3.22** P6 12.68 14.85 3.79** P9 TL3 52.98 61.30 3.33** TL6 40.15 47.40 3.71** TL9 AR3 1.12 1.12 .14 AR6 2.16 2.19 1.03 AR9 CI3 47.22 47.18 .16 CI6 31.67 31.39 1.20 CI9 MI3 47.48 54.92 3.08** MI6 18.54 21.63 3.77** MI 9 RL3 129 131 2.11* RL6 98 101 6.57** RL9 * = p<0 .05 ** = p<0 .01 CDF IDF T-VALUE VARIABLE CDF IDF T-VALUE 26.13 30.20 3.20** 11.92 13.92 3.44** 38.05 44.13 3.30** 2.19 2.17 .79 31.36 31.55 .86 17.38 20.36 3.48** 93 94 1.87 25.05 28.60 2.85** 11.43 13.13 3.07** 36.48 41.73 2.94** 2.19 2.18 .24 31.42 31.45 .10 16.66 19.16 3.07** 89 89 .56 23.93 26.98 2.64** 10.75 12.35 3.11** 34.68 39.33 2.80** 2.23 2.19 1.71 31.02 31.42 1.67 15.59 18.02 3.22** 85 84 1.62 Q10 22.45 25.00 2.44* P10 10.25 11.48 2.54* TL10 32.70 36.48 2.49* AR10 2.19 2.19 .04 CI10 31.43 31.40 .09 MI10 14.95 16.76 2.53* RL10 80 78 3.22** Q l l 20.88 23.25 2.44* P l i 9.58 10.70 2.65** T L l l 30.45 33.95 2.53* A R l l 2.18 2.17 .23 CI11 31.51 31.55 .14 M i l l 13.98 15.64 2.65** R L l l 74 73 2.72** Q12 28.57 31.75 2.42* TL12 28.57 31.75 2.42* RL12 70 68 3.34** Q13 22.98 25.08 1.98 TL13 22.98 25.08 1.98 RL13 56 54 5.64** 49 4.3. C o m p a r i s o n of t h e n=12 Spec i e s by A n a l y s i s o f V a r i a n c e An a n a l y s i s o f v a r i a n c e was p e r f o r m e d c o m p a r i n g e a c h c h a r a c t e r i s t i c of e v e r y chromosome f o r t h e n=l2 s p e c i e s . A Duncan's M u l t i p l e Range T e s t (DMRT) was e x e c u t e d on t h e measurement means t o d e t e r m i n e w h i c h s p e c i e s were s i g n i f i c a n t l y d i f f e r e n t f o r e a c h 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 . 4.3.1. Long Arm L e n g t h The r e s u l t s of t h e DMRT f o r t h e mean v a l u e s o f Q a r e p r e s e n t e d i n T a b l e 12. I t can be n o t e d t h a t chromosomes 1 and 3 a r e s e p a r a t e d i n t o two d i s t i n c t g r o u p s ; g r o u p one i n c l u d e s PM, PW and PF, g r o u p two c o n s i s t s of PG, PJ and PS. Chromosomes 2, 4, 5 and 6 a r e s e p a r a t e d i n t o t h r e e o v e r l a p p i n g g r o u p s . Chromosome 7 s e p a r a t e s t h e s p e c i e s i n t o 4 o v e r l a p p i n g c l a s s e s . Chromosomes 8, 9, 10 a n d 12 d i v i d e t h e s p e c i e s i n t o two o v e r l a p p i n g g r o u p s where PJ i s s i g n i f i c a n t l y d i f f e r e n t from PF i n chromosomes 8, 9 and 12, and f r o m PW i n chromosomes 8, 10 and 12. The Q v a l u e s f o r chromosome 11 do not d i f f e r s i g n i f i c a n t l y f o r any o f t h e s p e c i e s . 4.3.2. S h o r t Arm L e n g t h B a s e d on t h e m e t a c e n t r i c chromosomes (1 t o 6 ) , t h e m o r p h o m e t r i c v a l u e P d i v i d e s t h e s p e c i e s i n t o two g r o u p s ( T a b l e 13). The f i r s t g r o u p i n c l u d e s PM, PF and PW w h i l e t h e s e c o n d g r o u p c o n t a i n s P J , PG and PS. Chromosomes 7, 9 and 10 a l s o s e p a r a t e t h e s p e c i e s i n t o two c l a s s e s , b u t d i f f e r from t h e 50 p r e v i o u s chromosomes i n t h a t PM i s c l a s s i f i e d i n t h e s e c o n d g r o u p w i t h P J , PG and PS. Chromosomes 8 and 12 d i v i d e t h e s p e c i e s i n t o t h r e e g r o u p s , one d i s t i n c t (PF and PW) g r o u p and two o v e r l a p p i n g g r o u p s (PG, P J , PM and P S ) . F o r chromosome 8, PG (15.15) i s s i g n i f i c a n t l y d i f f e r e n t from PS (16.43) but n e i t h e r o f t h e s e d i f f e r s i g n i f i c a n t l y from PJ (15.53) o r PM ( 1 5 . 8 0 ) . PG (11.70) d i f f e r s from PM (12.60) f o r chromosome 12, w h i l e n e i t h e r d i f f e r s i g n i f i c a n t l y f r o m PJ (11.88) or PS ( 1 2 . 4 8 ) . Chromosome 11 d i v i d e s t h e s p e c i e s i n t o f o u r o v e r l a p p i n g g r o u p s . 4.3.3. T o t a l L e n g t h Chromosomes 1 t o 5 s e p a r a t e t h e s i x s p e c i e s i n t o two s i g n i f i c a n t g r o u p s b a s e d on t h e a v e r a g e TL ( T a b l e 1 4 ) . The f i r s t g r o u p c o n s i s t s o f PM, PF and PW w h i l e t h e s e c o n d - g r o u p i n c l u d e s PG, PJ and PS. The TL of chromosome 6 d i v i d e s t h e s p e c i e s i n t o t h r e e o v e r l a p p i n g c l a s s e s . Chromosomes 7 t o 12 s e p a r a t e t h e s p e c i e s i n t o two o v e r l a p p i n g g r o u p s . F o r chromosome 7, PM (52.43) i s i n t h e o v e r l a p r e g i o n i n d i c a t i n g t h a t i t i s not s i g n i f i c a n t l y d i f f e r e n t f r o m any of t h e o t h e r s p e c i e s . PF (48.30) and PW (48.65) a r e s i g n i f i c a n t l y d i f f e r e n t f r o m PG ( 5 3 . 9 3 ) , P J (55.33) and PS ( 5 5 . 9 3 ) . PM and PG a r e i n t h e o v e r l a p r e g i o n s f o r chromosomes 8 t o 11, where PF and PW d i f f e r from PS and P J . F o r chromosome 12, PG i s i n t h e o v e r l a p r e g i o n , w h i l e PF and PW d i f f e r s i g n i f i c a n t l y from PM, PS and P J . TABLE 12: DUNCAN'S MULTIPLE RANGE TEST LONG ARM LENGTH* (Oj CHROMOSOME 10 PM PW PF PG PJ PS 37.20 38.75 38.91 43.75 43.38 44.35 PM PW PF PG' PJ PS 35.13 36.90 38.15 40.10 40.88 41.68 PM PF PW PJ PG PS 33.73 34.15 35.28 38.90 39.28 39.78 PF PM PW PG PJ PS 31.91 32.58 34.33 36.73 37.10 37.75 PM PF PW PG PJ PS 31.58 32.25 32.95 35.33 35.73 36.48 PF PM PW PG PJ PS 29.25 30.60 31.53 33.03' 34.25 34.73 PW PF PM PG PS PJ 34.13 34.25 35.55 37.50 38.20 38.63 PW PF PM PS PG PJ 31.65 31.80 33.60 34.35 34.35 35.93 PF PW PM PG PS PJ 29.90 30.18 31.75 32.63 32.65 33.93 PW PF PM PG PS PJ 28.78 28.95 29.92 30.88 31.05 32.03 H PF PW PM PS PG PJ 27.00 27.18 27.85 29.05 29.35 30.03 12 pp PW PM PG PS PJ 24.30 24.45 26.45 26.65 26.70 27.33 * map measure units: 46 units = 10 ym TABLE 1 3 : DUNCAN'S MULTIPLE RANGE TEST SHORT ARM LENGTH* (P) CHROMOSOME PM PF PW P J PG PS 3 4 . 7 5 3 5 . 5 0 3 5 . 8 5 4 0 . 9 5 4 1 . 1 8 4 2 . 0 3 PF PM PW P J PG PS 3 0 . 4 0 3 2 . 6 5 3 3 . 5 5 3 8 . 3 8 3 8 . 5 5 3 9 . 1 8 PM PF PW PG P J PS 3 1 . 3 3 3 1 . 3 5 3 2 . 5 5 3 6 . 6 0 3 6 . 8 8 3 7 . 1 3 PM PF PW PG P J PS 3 0 . 3 2 3 1 . 3 0 3 1 . 4 8 3 5 . 2 8 3 5 . 6 5 3 5 . 9 8 PF PM PW PG P J PS 2 8 . 6 5 2 9 . 2 0 3 0 . 1 5 3 3 . 7 5 3 4 . 0 8 3 4 . 2 5 6 PM PF PW PG PS P J 2 8 . 0 3 2 8 . 3 5 2 8 . 9 3 3 1 . 9 3 3 2 . 6 3 3 2 . 9 2 PF PW PG P J PM PS 1 4 . 0 5 1 4 . 5 3 1 6 . 4 3 1 6 . 7 0 1 6 . 8 8 1 7 . 7 3 8 PF PW PG P J PM PS 1 2 . 9 0 1 3 . 3 5 1 5 . 1 5 1 5 . 5 3 1 5 . 8 0 1 6 . 4 3 PW PF PG P J PM PS 1 2 . 5 8 1 2 . 6 5 1 4 . 4 0 1 4 . 4 0 1 5 . 0 0 1 5 . 3 0 10 PF PW P J PG PM PS 1 1 . 9 5 1 2 . 1 0 1 3 . 5 6 1 3 . 6 0 1 4 . 0 3 1 4 . 4 5 11 PW PF PG P J PM PS 1 1 . 3 8 1 1 . 5 0 1 2 . 6 5 1 2 . 9 5 1 3 . 4 8 1 3 . 8 8 12 PW PF PG P J PS PM 1 0 . 7 0 1 0 . 7 5 1 1 . 7 0 1 1 . 8 8 1 2 . 4 8 1 2 . 6 0 * map measure u n i t s : 46 u n i t s •> 10 urn TABL3 1 4 : DUNCAN'S MULTIPLE RANGE TEST TOTAL LENGTH* (TL) CHROMOSOME PM PF PW P J PG PS 7 1 . 9 5 7 4 . 4 0 7 4 . 6 0 8 4 . 3 2 8 4 . 4 0 6 6 . 3 4 PM PF PW PG P J PS 6 7 . 7 8 6 8 . 5 5 7 0 . 4 5 7 8 . 6 5 7 9 . 2 5 8 0 . 8 5 PM PF PW PG PJ PS 6 5 . 0 5 6 5 . 5 0 6 7 . 8 3 7 4 . 8 8 7 5 . 7 8 7 6 . 9 0 PM PF PW PG P J PS 6 2 . 9 0 6 3 . 2 5 6 5 . 7 0 7 2 . 0 0 7 2 . 7 5 7 3 . 7 3 PM PF PW PF P J PS 6 0 . 7 8 6 0 . 9 0 6 3 . 1 0 6 9 . 0 8 7 2 . 7 5 7 0 . 7 3 PF PM PW PG P J PS 5 7 . 6 0 5 8 . 6 3 6 3 . 1 0 6 4 . 9 5 6 7 . 1 8 6 7 . 3 5 PF PW PM PG P J PS 4 8 . 3 0 4 8 . 6 5 5 2 . 4 3 5 3 . 9 3 5 5 . 3 3 5 5 . 9 3 PF PW PM PG PS P J 4 4 . 7 0 4 5 . 0 0 4 9 . 4 0 4 9 . 5 0 50 .74 5 1 . 4 5 9 PF PW PM PG PS P J 4 2 . 5 5 4 2 . 7 5 4 6 . 7 5 4 7 . 0 3 4 7 . 9 5 4 8 . 3 3 10 PW PF PM PG PS P J 4 0 . 8 8 4 0 . 9 0 4 3 . 9 5 4 4 . 4 8 4 5 . 4 8 4 5 . 6 0 11 PF PW PM PG PS P J 3 8 . 5 0 3 8 . 5 5 4 1 . 3 3 4 2 . 0 0 4 2 . 9 3 4 2 . 9 8 12 PF PW PG PM PS P J 3 5 . 0 5 3 5 . 1 5 3 8 . 3 5 3 9 . 2 0 3 9 . 1 8 3 9 . 2 0 * map measure u n i t s : 46 u n i t s * 10 nm 54 4.3.4. Arm R a t i o The i n d e x v a l u e AR d i v i d e s t h e s p e c i e s i n t o d i f f e r e n t s i g n i f i c a n t g r o u p s as i s p r e s e n t e d i n T a b l e 15. The AR of chromosome 1 s e p a r a t e s t h e s p e c i e s i n t o f o u r o v e r l a p p i n g g r o u p s . The AR o f chromosomes 2 and 8 d i v i d e t h e s p e c i e s i n t o f o u r d i s t i n c t c l a s s e s . F o r chromosome 2, PG ( 1 . 0 4 ) , PW (1.10) a n d ~ P F (1.25) e a c h b e l o n g t o s e p a r a t e c l a s s i f i c a t i o n g r o u p s , hence e a c h a r e s i g n i f i c a n t l y d i f f e r e n t from t h e o t h e r f i v e s p e c i e s . PS ( 1 . 0 7 ) , P J (1.07) and PM (1.08) b e l o n g t o t h e same g r o u p . F o r chromosome 8, PW (2.37) and PF (2.49) b e l o n g t o s e p a r a t e g r o u p s w h i l e PG (2.27) and PJ (2.31) s h a r e a g r o u p and PS (2.09) and PM (2.13) s h a r e a n o t h e r g r o u p . Chromosomes 7 and 9 s e p a r a t e t h e s p e c i e s i n t o t h r e e d i s t i n c t g r o u p s . PF b e l o n g s t o one g r o u p , w h i l e PM and PS, and PG, PJ and PW f o r m t h e o t h e r two g r o u p s . PG i s t h e s i n g l e member o f a g r o u p f o r chromosome 9, w h i l e PM and PS, and P J , PF and PW c o m p r i s e t h e o t h e r two g r o u p s . Chromosomes 3 and 12 d i v i d e t h e s p e c i e s i n t o two s e p a r a t e g r o u p s . Chromosomes 4, 5, 10 and 11 s e p a r a t e t h e s p e c i e s i n t o t h r e e o v e r l a p p i n g g r o u p s and chromosome 6 s p l i t s t h e s p e c i e s i n t o two - o v e r l a p p i n g g r o u p s . 4.3.5. C e n t r o m e r e Index The most d i s c r i m i n a t i n g chromosomes b a s e d on CI v a l u e s ( T a b l e 16) a r e chromosomes 2 and 8, where t h e s p e c i e s a r e s e p a r a t e d i n t o f o u r d i s t i n c t g r o u p s . The CI v a l u e of chromosome 2 55 p l a c e s PF ( 4 4 . 4 1 ) , PW (47.71) and PG (48.96) i n t o s e p a r a t e g r o u p s w h i l e PM ( 4 8 . 1 8 ) , PJ (48.40) and PS (48.42) c o n s t i t u t e t h e f o u r t h g r o u p . Chromosome 8 a s s i g n s PF (29.11) and PW (29.70) t o i n d i v i d u a l c l a s s e s , P J (30.21) and PG (30.61) c o m p r i s e t h e t h i r d g r o u p w h i l e PM (31.99) and PS (32.27) a r e i n c l u d e d i n t h e f o u r t h g r o u p . Chromosomes 7, 9 and 10 s e p a r a t e t h e s p e c i e s i n t o t h r e e n o n - o v e r l a p p i n g g r o u p s where t h e CI v a l u e f o r chromosome 7 o f PF and chromosomes 9 and 10 of PG a r e s i g n i f i c a n t l y d i f f e r e n t (p<0.05) f r o m t h e o t h e r s p e c i e s . Chromosome 5 d i v i d e s t h e s p e c i e s i n t o f o u r g r o u p s , w i t h g r o u p one c o n s i s t i n g of PF, w h i l e t h e r e m a i n i n g t h r e e g r o u p s o v e r l a p . Chromosomes 1 , 3 , 4, 6 and 11 a r r a n g e t h e s p e c i e s i n t o two t o f o u r o v e r l a p p i n g g r o u p s . Chromosome 12 d i v i d e d t h e s p e c i e s i n t o two g r o u p s . 4.3.6. M o r p h o l o g i c a l Index The most d i s c r i m i n a t i n g MI v a l u e s b e l o n g t o chromosome 2, a s i s e v i d e n t i n T a b l e 17. The MI v a l u e s of chromosome 2 a r e s e p a r a t e d i n t o t h r e e s i g n i f i c a n t g r o u p s ; g r o u p one i n c l u d e s PF, g r o u p two c o n t a i n s PM and PW w h i l e g r o u p t h r e e c o n s i s t s o f P J , PG and PS. Chromosomes 1, 3, 4 and 5 d i v i d e t h e s p e c i e s i n t o two g r o u p s where t h e f i r s t g r o u p c o n t a i n s PM, PF and PW and t h e s e c o n d g r o u p i n c l u d e s PG, PJ and PS. Chromosomes 7, 8, 9 and 10 s e p a r a t e t h e s p e c i e s i n t o one d i s t i n c t and two o v e r l a p p i n g g r o u p s . Chromosome 6 d i v i d e s t h e s p e c i e s i n t o t h r e e o v e r l a p p i n g g r o u p s . Chromosome 11 s e p a r a t e s them i n t o 3 d i f f e r e n t g r o u p s w h i c h i n c l u d e s PW and PF i n g r o u p one, PG and PJ i n g r o u p two and PM and PS i n t h e t h i r d g r o u p . Chromosome 12 s e p a r a t e s t h e s p e c i e s i n t o one u n i q u e and t h r e e o v e r l a p p i n g g r o u p s . CHROMOSOME TABLE 1 5 : DUNCAN'S MULTIPLE RANGE TEST ARM RATIO (AR) PG PS P J PC PW PF 1.05 1.06 1.06 1.07 1.08 1.10 2 4 10 11 12 PG PS PJ PM PW PF 1.04 1.07 1.07 1.08 1.10 1.25 PG P J PS PM PW PF 1.05 1.06 1.07 1.08 1.08 1.09 PF P J PG PS PM PW 1.02 1.04 1 .05 1 .05 1 .08 1 .09 P J PG PS PM PW PF 1.05 1.05 1.07 1 .08 1 .09 1.13 PF PG P J PS PW PM 1.03 1.03 1 .06 1.07 1 .09 1 .09 PM PS PG P J PW PF 2 . 1 1 2 . 1 6 2 . 2 9 2 . 3 2 2 . 3 5 2 . 4 5 PS PM PG P J PW PF 2 . 0 9 2 . 1 3 2 .27 2 . 3 1 2 . 3 7 2 . 4 9 PM PS PG P J PW PF 2 . 1 2 2 . 1 5 2 . 2 7 2 . 3 6 2 . 3 8 2 . 4 0 PM PS PG P J PW PF 2 . 1 4 2 . 1 5 2 . 2 8 2 . 3 6 2 . 3 7 2 . 4 4 PM PS PG P J PF PW 2 . 0 7 2 . 1 0 2 . 3 2 2 . 3 2 2 . 3 9 2 . 3 9 PM PS PF PG PW P J 2.10 2.14 2.26 2.28 2.29 2.30 56 TABLE 16: DUNCAN'S MULTIPLE RANGE TEST CENTROMERE INDEX (CI) CHROMOSOME PF PW PM PJ PS PG 47.68 48.14 48.23 48.56 48.67 48.79 PF PW PM PJ PS PG 44.41 47.71 48.18 48.40 48.42 48.96 pp PW PM PS PJ PG 47.83 48.01 48.16 48.27 48.68 48.90 PW PM PS PG PJ PF 47.91 48.21 48.81 48.92 49.00 49.46 PF PW PM PS PG PJ 47.08 47.78 48.07 48.43 48.77 48.81 6 PM PW PS PJ PG PF 47.85 47.91 48.44 48.81 49.17 49.24 PF PW PJ PG PS PM 29.11 29.92 30.20 30.14 31.70 32.18 PF PW PJ PG PM PS 28.77 29.70 30.21 30.61 31.99 32.37 9 PW PF PJ PG PS PM 29.46 29.69 29.83 30.58 31.82 32.09 10 PF PW PJ PG PS PM 29.17 29.74 29.77 30.55 31.85 31.91 11 PW PF PJ PG PS PM 29.61 29.91 30.14 30.18 32.36 32.66 57 12 PW PJ PG PF PS PM 30.50 30.52 30.52 30.75 31.90 32.30 TABLE 17: DUNCAN'S MULTIPLE RANGE TEST MORPHOLOGICAL INDEX (MI) CHROMOSOME 10 11 12 PM PF PW PJ PG PS 67.25 67.91 69.10 79.68 80.42 81.94 PF PM PW PJ PG PS 54.70 63.03 64.13 74.45 75.64 76.05 PF PM PW PG PS PJ 60.17 60.45 62.63 71.63 71.85 71.86 PM PW PF PG PJ PS 58.60 60.51 61.98 69.20 69.93 70.29 PF PM PW PG PS PJ 54.14 56.23 57.80 66.03 66.46 66.62 PM PW PF PJ PG PS 53.74 55.53 55.85 60.70 62.81 63.31 p F pw PG PJ PM p s 19.85 20.74 23.64 23.95 24.91 25.97 R PF PW PG PJ PM PS . 18 17 19 00 21.84 22.25 23.24 24.30 PW PF PJ PG PM PS 17.83 18.04 20.53 20.77 22.10 22.51 PF PW PJ PG PM PS 16.91 17.21 19.34 19.60 20.61 21.21 PW PF PG PJ PM PS 16.16 16.91 18.12 18.55 20.01 20.53 PW PF PG PJ PS PM 15.41 15.52 16.85 17.05 18.33 18.61 59 4.3.7. R e l a t i v e L e n g t h The most d i s c r i m i n a t i n g chromosomes b a s e d on RL a r e 1 and 3 ( T a b l e 18). Chromosome 1 d i v i d e s t h e s p e c i e s i n t o f o u r g r o u p s , two of which o v e r l a p . PM (131) and PW (137) a r e s i g n i f i c a n t l y d i f f e r e n t (p<0.05) from a l l o t h e r s p e c i e s . Chromosome 3 p l a c e s PM (118) i n t o one g r o u p and PF (123) i n t o a n o t h e r , b o t h of w h i c h a r e s i g n i f i c a n t l y d i f f e r e n t (p<0.05) from a l l o t h e r s p e c i e s . The RL of PM i s s i g n i f i c a n t l y d i f f e r e n t (p<0.05) from t h e r e m a i n i n g s p e c i e s f o r a l l chromosomes e x c e p t chromosome 6. Chromosome 2, 4 and 11 s e p a r a t e t h e s p e c i e s i n t o f o u r g r o u p s , PM and t h r e e o v e r l a p p i n g g r o u p s . Chromosome 6 d i v i d e s t h e s p e c i e s i n t o 3 o v e r l a p p i n g g r o u p s . Chromosomes 5, 7, 8, 9, 10 and 12 s e p a r a t e t h e s p e c i e s i n t o t h r e e g r o u p s , PM and two o v e r l a p p i n g g r o u p s . 4.4. C o m p a r i s o n of 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 (n-13) by  M u l t i v a r i a t e A n a l y s i s A m u l t i v a r i a t e d i s c r i m i n a n t f u n c t i o n a n a l y s i s ( J e n n r i c h and Sampson, 1981) i s u s e d t o p r o v i d e a n u m e r i c a l a n a l y s i s f o r 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 (n=13) and f o r t h e r e m a i n i n g s i x s p e c i e s w i t h n=l2 chromosomes. T h i s a n a l y s i s m a x i m i z e s t h e v a r i a t i o n among s p e c i e s r e l a t i v e t o t h e v a r i a t i o n w i t h i n s p e c i e s . The v a r i a b l e s u s e d i n c o m p u t i n g t h e l i n e a r c l a s s i f i c a t i o n f u n c t i o n s a r e c h o s e n i n a s t e p w i s e manner. At e a c h s t e p , t h e v a r i a b l e t h a t adds t h e most t o t h e s e p a r a t i o n o f t h e s p e c i e s i s e n t e r e d i n t o t h e d i s c r i m i n a n t f u n c t i o n . TABLE 18: DUNCAN'S MULTIPLE RANGE TEST RELATIVE LENGTH (RL) CHROMOSOME 1 PM PW PJ PF PS PG 131 137 138 139 140 141 2 PM PF PW PJ PG PS 123 129 129 130 131 132 3 PM PF PJ PS PG PW 118 123 124 125 125 125 PM PF PJ PS PG PW 114 119 119 120 120 121 PM PF PG PS PJ PW 111 114 115 115 115 116 PW PG PF PJ PS PM 107 108 108 109 110 111 PW PG PF PJ PS PM 69 90 91 91 91 95 PG PS PW PF PJ PM 83 83 83 84 84 90 PS PG PW PJ PF PM 78 78 79 79 80 85 PS PG PJ PW PF PM 74 74 75 75 77 80 PS PG PJ PW PF PM 70 70 71 71 72 75 PS PG PJ PW PF PM 64 64 64 65 66 71 61 The d i s c r i m i n a n t a n a l y s i s u s e d t o s e p a r a t e t h e two v a r i e t i e s u t i l i z e d s e v e n v a r i a b l e s , RL6, RL1, MI 9, MI 5, RL3, RL11 and RL13, ( T a b l e 1 9 ) . The two v a r i e t i e s ' means (F=13.53) a r e s i g n i f i c a n t l y d i f f e r e n t ( p < 0 . 0 l ) from e a c h o t h e r . The j a c k - k n i f e d c l a s s i f i c a t i o n p r o c e d u r e y i e l d e d an 86.2% c o r r e c t c l a s s i f i c a t i o n f o r t h e d i f f e r e n t k a r y o t y p e s ( T a b l e 19), where 34 of t h e 40 CDf k a r y o t y p e s were a s s i g n e d t o t h e CDf c l a s s i f i c a t i o n g r o u p and 35 of t h e 40 IDf k a r y o t y p e s were a s s i g n e d t o t h e IDf g r o u p . A h i s t o g r a m of t h e c a n o n i c a l v a r i a b l e s e p a r a t i n g t h e 80 D o u g l a s - f i r k a r y o t y p e s ( F i g u r e 11) d e m o n s t r a t e s t h a t t h e two v a r i e t i e s c a n be s e p a r a t e d . T h e r e i s some o v e r l a p between t h e CDf samples (A) and t h e IDf samples ( B ) . 4.5. C o m p a r i s o n of t h e n=12 S p e c i e s by M u l t i v a r i a t e A n a l y s i s F o u r t e e n v a r i a b l e s a r e i n c l u d e d i n t h e d i s c r i m i n a n t a n a l y s i s o f t h e s i x s p e c i e s . The v a r i a b l e s a r e AR2, RL12, CI11, RL8, C I 8 , AR4, RL1, C I 7 , AR6, RL7, C I 9 , AR11, AR5 and CI10 ( T a b l e 2 0 ) . A l l s p e c i e s means a r e s i g n i f i c a n t l y d i f f e r e n t a t t h e 1% l e v e l ( p < 0 . 0 l ) w i t h he e x c e p t i o n of PG and PJ w h i c h a r e s i g n i f i c a n t l y d i f f e r e n t a t t h e 5% l e v e l ( T a b l e 2 0 ) . The j a c k - k n i f e d c l a s s i f i c a t i o n m a t r i x ( T a b l e 20) p l a c e s 85% of t h e PF k a r y o t y p e s i n t h e c o r r e c t c l a s s i f i c a t i o n g r o u p , 80% of th e PS s a m p l e s , 70% o f t h e PG s a m p l e s , 55% of t h e P J s a m p l e s , 70% of t h e PW samp l e s and 97.5 of t h e PM k a r y o t y p e s . T h e r e i s c o n s i d e r a b l e o v e r l a p between PG and P J as i s e v i d e n t by t h e f a c t 62 TABLE 19 Results of discriminant analysis of the two P. menziesii varieties Step Variable Coefficient for Variable Symbol1 Entered canonical variable Number 1 -21.557 42 RLg 2 -11.562 7 RL! 3 0.110 62 MIg 4 -35.388 • 34 ML5 5 - 0.427 21 RL3 6 -14.548 77 RL n 7 14.456 91 RL 1 3 Constant 86.498 Eigenvalue: 1.320. Cumulative proportion of total dispersion: 1.0. Canonical correlation: 0.75. Matrix of F value for testing group means: df = 7,72tt CDf IDf 13.53 Jack-Tcnifed Classification Matrix: Number Classified As:  Percentage Group CDf IDf Correct CDf 34 6 85.0 IDf 5 35 87.5 86.2% of calibration group correctly classified to source species. t t t See Materials and Methods for explanation of symbols. Significance levels for F-values at 7,72 df are: a = 0.05, F = 2.15; a = 0.01, F = 2.90. B B B BB B B A A B BB B B BBB B A A A . AA A A AA B B B BBB BB BBBB BB BAR B BBBAABBBAAAAAAA AAAAAAAA A A AAA BA A AAA A .. + .... + .... + .... + .... + .... + .... + ... .+2. .. + .... + .... + .... + .... + ...1+. ...+....+... . + .... + .... + .... + .... -4.0 -3.2 -2.4 -1.6 -.80 0.0 .80 1.6 2.4 3.2 4 -3.6 -2.8 -2.0 -1.2 -.40 .40 1.2 2.0 2.8 3.6 FIGURE 11: Histogram of the Canonical Variable of CDf (A) and IDf (B) 64 TABLE 20 Results of discriminant analysis of the s i x Pseudotsuga species (n=12) Step V a r i a b l e C o e f f i c i e n t s f o r canonical v a r i a b l e Variable Symbol+ Entered Number 1 2.789 - 4.924 2 4.740 16.690 3 - 0.744 - 2.472 4 2.035 4.750 5 - 2.475 - 0.317 6 - 0.255 - 0.117 7 -12.319 4.032 8 - 0.250 - 0.232 9 -12.055 4.140 10 - 0.196 0.029 11 - 0.142 0.094 12 7.600 4.701 13 0.506 0.506 14 -19.606 14.168 Constant 19.674 -49.215 15.937 11 AR 2 13.284 84 R L 1 2 - 9.471 75 cm - 0.619 56 RI+3 - 3.951 54 C I 8 0.059 25 AR4 9.721 7 RL! 0.332 47 C I 7 - 4.496 39 ARs 0.160 49 RL 7 0.098 61 CIg - 6.123 74 A R U - 0.355 32 AR5 -11.180 68 ciio -14.425 Eigenvalues: 5.075, 1.941, 1.358. Cumulative proportion of t o t a l dispersion: 0.56, 0.78, 0.93. Canonical c o r r e l a t i o n s : 0.91, 0.81, 0.77. Matrix o f F value f o r t e s t i n g group means: df = 14,201+''' PS 31.94 PG 25.30 11.56 PJ 21.85 15.52 2.16 PW 20.35 26.82 11.05 8.26 PM 52.47 29.56 42.87 40.61 51.98 PF PS PG PJ PW Jack-knifed C l a s s i f i c a t i o n Matrix: Number C l a s s i f i e d As:  Percentage Group PF PS PG PJ PW PM Correct PF 17 1 — — 2 — 85.0 PS — 32 3 2 — 3 80.0 PG — — 28 10 2 — 70.0 PJ — — 14 22 4 — 55.0 PW 1 1 4 6 28 — 70.0 PM — — 39 97.5 75.5% of c a l i b r a t i o n group c o r r e c t l y c l a s s i f i e d t o source species. See Materials and Methods f o r explanation of symbols. Si g n i f i c a n c e l e v e l s f o r F-values a t 14,201 d f are: a = 0.05, F = 1.74; a = 0.01, F = 2.17. 65 t h a t 10 of t h e 40 PG samples a r e c l a s s i f i e d a s P J and 14 of t h e 40 P J sam p l e s a r e c l a s s i f i e d a s PG. A t o t a l o f 75.5% of t h e c a l i b r a t i o n g r o u p s a r e c o r r e c t l y c l a s s i f i e d t o s o u r c e s p e c i e s . When t h e s p e c i e s means a r e p l o t t e d on c a n o n i c a l v a r i a b l e s 1, 2 and 3 ( F i g u r e 12), t h e f i r s t c a n o n i c a l v a r i a b l e , w h i c h a c c o u n t s f o r 56.4% o f t h e d i s p e r s i o n , s e p a r a t e s PM from t h e r e m a i n i n g f i v e s p e c i e s . PG, P J and PW a r e g r o u p e d c l o s e l y t o g e t h e r . C a n o n i c a l v a r i a b l e s 2 and 3, w h i c h a c c o u n t f o r 22.6% and 15.1% of t h e d i s p e r s i o n , s e p a r a t e s PW f r o m PG and P J . S i n c e PM i s so r e a d i l y d i s t i n g u i s h a b l e , i t i s p o s s i b l e t h a t some of t h e power of d i s c r i m i n a t i o n among t h e o t h e r s p e c i e s , p a r t i c u l a r l y between PG and PJ a r e l o s t i n t h e p r e s e r v a t i o n of t h e d i s t a n c e between PM and t h e o t h e r f i v e s p e c i e s . T h e r e f o r e , on t h e b a s i s o f t h i s a s s u m p t i o n a t w o - s t a g e a n a l y s i s was us e d w h i c h o m i t t e d PM and i n c l u d e d o n l y t h e f i v e A s i a t i c s p e c i e s . F o u r t e e n v a r i a b l e s a r e i n c l u d e d i n t h i s s e c o n d d i s c r i m i n a n t a n a l y s i s , AR2, C I 8 , CI 11 , AR4, RL1, MI 1, Q9, P10, CI 1, AR6,.RL8, MI8, MI 3 and P6 ( T a b l e 2 1 ) . A l l s p e c i e s means a r e s i g n i f i c a n t l y d i f f e r e n t ( p < 0 . 0 l ) f r o m e a c h o t h e r . In t h e j a c k - k n i f e d c l a s s i f i c a t i o n m a t r i x ( T a b l e 2 1 ) , 73.3% o f t h e d i f f e r e n t k a r y o t y p e s a r e c o r r e c t l y i d e n t i f i e d t o s p e c i e s . PF has 85% of i t s k a r o t y p e s c o r r e c t l y c l a s s i f i e d , PS has 92.5%, PG has 65.0%, P J has 52.5% and PW has 77.5% o f t h e s a m p l e s c o r r e c t l y c l a s s i f i e d . The t h r e e d i m e n s i o n a l p l o t ( F i g u r e 13) shows t h a t t h e t h r e e c a n o n i c a l v a r i a b l e s , w h i c h a c c o u n t f o r 48.5, 38.2 and 10.3%, r e s p e c t i v e l y o f t h e t o t a l d i s p e r s i o n , s e p a r a t e t h e f i v e s p e c i e s from e a c h o t h e r . CANONICAL VARIABLE 1 (56.4%) FIGURE 12: Plot of the n=12 species means as evaluated at each of the f i r s t three canonical variables obtained by discriminant analysis of karyotype numerical data. Percentage di s p e r s i o n associated with each canonical y a r i a t e shown i n parentheses and also used to determine r a t i o of axes length. 67 TABLE 21 Results of discriminant analysis of the five Asiatic species Step Variable Coefficients for canonical variable Variable Symbol''' Entered Number 1 14.022 17.515 2 - 0.763 - 0.544 3 - 0.240 0.363 4 - 1.216 - 4.555 5 - 3.967 2.622 6 - 0.230 0.111 7 - 0.037 - 0.584 8 0.001 0.346 9 - 0.312 - 0.171 10 - 1.981 - 4.688 11 -13.264 -18.062 12 0.426 0.998 13 - 0.045 - 0.090 14 - 0.031 - 0.107 Constant 51.752 15.537 Eigenvalues: 2.970, 2.340, 0.630. Cumulative proportion of to t a l dispersion: 0.49, Canonical correlations: 0.86, 0.84, 0.62. Matrix of F value for testing group means: df = 1.116 11 ' AR2 0.440 54 C I 8 - 0.077 75 c i i i -13.472 25 AR4 16.255 7 - 0.018 48 MI7 0.221 57 09 - 0.271 65 PlO 0.200 5 C l ! - 9.014 39 ARg 26.496 56 RLQ - 0.441 57 MI 8 0.141 20 MI 3 - 0.078 37 ?6 -43.732 0.87, 0.97. 14/162t"t' PS 30.72 PG 28.00 15.61 PJ 25.29 18.99 2.58 PW 20.51 26. "55 10.02 7.03 PF PS PG PJ Jack-knifed Classification Matrix: Number Classified As:  Percentage Group PF PS PG PJ PW Correct PF 17 1 1 — 1 85.0 PS — 37 1 — 2 92.5 PG — — 26 10 4 65.0 PJ — 1 14 21 4 52.5 PW — 1 2 6 31 77.5 73.3% of calibration group correctly c l a s s i f i e d to source species. * See Materials and Methods for explanation of symbols. ft Significance levels for F-values at 14,162 df are : a = 0.05, F = 1.76, a •= 0.01, F = 2.19 o O -4.0 - 2 . 0 0 2 0 ' 4 0 CANONICAL VARIABLE 1 (48%) FIGURE 13: P l o t o f t h e A s i a t i c s p e c i e s means as e v a l u a t e d a t e a c h o f t h e f i r s t t h r e e c a n o n i c a l v a r i a b l e s o b t a i n e d b y d i s c r i m i n a n t a n a l y s i s o f k a r y o t y p e n u m e r i c a l d a t a . P e r s e n t a g e d i s p e r s i o n a s s o c i a t e d w i t h each c a n o n i c a l v a r i a t e shown i n p a r e n t h e s e s and a l s o u s e d t o d e t e r m i n e r a t i o o f axes l e n g t h s . co 69 5. DISCUSSION The s p e c i e s i n t h e P s e u d o t s u g a genus a r e d i v i d e d i n t o two g r o u p s b a s e d on chromosome number. P_j_ m e n z i e s i i , more commonly r e f e r r e d t o as D o u g l a s - f i r c o n t a i n s 26 s o m a t i c chromosomes w h i l e t h e r e m a i n i n g s p e c i e s , P^ m a c r o c a r p a , P. f o r r e s t i i , P. s i n e n s i s , P. j a p o n i c a , P. w i l s o n i a n a and P_;_ g a u s s e n i i have a s o m a t i c chromosome number of 24. D i f f e r e n c e s were o b s e r v e d i n t h e l o n g and s h o r t arm l e n g t h s f o r s e v e r a l chromosomes, w h i c h s u b s e q u e n t l y a f f e c t e d t h e c a l c u l a t e d p a r a m e t e r s ( T L , AR, C I , MI and R L ) . G i v e n t h e l a r g e number of v a r i a b l e s i n v o l v e d ; 84 f o r n=l2 s p e c i e s , 91 f o r n=13 s p e c i e s , any s u b j e c t i v e i n t e r p r e t a t i o n of t h e s e d i f f e r e n c e s would be i n a d e q u a t e f o r d e t e c t i n g s l i g h t s p e c i e s v a r i a t i o n s . The t -t e s t , a n a l y s i s o f v a r i a n c e and m u l t i v a r i a t e a n a l y s i s were a b l e t o d e t e c t m i n o r , but s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s between t h e s p e c i e s and v a r i e t i e s . Q, P and TL were i n c l u d e d i n t h e v a r i o u s s t a t i s t i c a l a n a l y s e s b e c a u s e t h e o t h e r v a r i a b l e s were b a s e d on t h e s e measurements, but t h e s e v a l u e s w i l l not be i n c l u d e d i n t h e d i s c u s s i o n . The l e n g t h s o f t h e chromosomes v a r i e d f r o m one c e l l t o a n o t h e r w i t h i n s p e c i e s d e p e n d i n g on t h e m i t o t i c s t a g e o f t h e c e l l a t f i x a t i o n . J a c k s o n (1971) n o t i c e d t h a t w i t h i n i n d i v i d u a l r o o t t i p p r e p a r a t i o n s c o n s i d e r a b l e v a r i a t i o n e x i s t e d w h i c h may be c a u s e d by d i f f e r e n t i a l p e n e t r a t i o n o f f i x i n g and h y d r o l y z i n g c h e m i c a l s so t h a t t h e o u t e r c e l l s may have p r o l o n g e d t r e a t m e n t s . G e n e r a l l y t h e b e s t s p r e a d s were t h o s e t h a t c o n t a i n e d v e r y 70 c o n d e n s e d , h i g h l y c o i l e d chromosomes. S i n c e t h e r e was a d e f i n i t e t e n d e n c y t o s e l e c t t h e s e c e l l s f o r measurements, Q, P and TL v a l u e s were n o t random but skewed t o w a r d s t h e s m a l l e r more c o n d e n s e d chromosomes. On t h e o t h e r hand, AR, C I , MI and RL a r e i n d e p e n d e n t of t r e a t m e n t c o n d i t i o n s o r m i t o t i c s t a g e a t f i x a t i o n , so a l l d i s c u s s i o n s c o n c e r n i n g v a r i a t i o n s among s p e c i e s a r e b a s e d on t h e s e v a l u e s . 5.1. Chromosome Number A c o n s i s t e n t h a p l o i d chromosome number o f n=13 was f o u n d i n a l l c e l l s examined f o r t h e two v a r i e t i e s o f D o u g l a s - f i r , w h i c h i n c l u d e d f i v e m e t a c e n t r i c , s i x s u b m e t a c e n t r i c and two t e l o c e n t r i c p a i r s . The chromosome numbers a g r e e w i t h t h e o b s e r v a t i o n s o f C h r i s t i a n s e n .(1963), Thomas and C h i n g ( 1 9 6 8 ) , L i v i n g s t o n ( 1 9 7 1 ) , D e V e s c o v i and S z i k l a i (1975) and Wochok e t a l . ( 1 9 8 0 ) . R e p o r t s o f a chromosome number of n=l2 f o r D o u g l a s - f i r have been made ( L a n g l e t , 1934; D u r r i e u - V a b r e , 1958) but have a l w a y s been q u e s t i o n e d by o t h e r a u t h o r s . R are chromosomal a b e r r a t i o n s have a l s o been r e v e a l e d f o r t h i s s p e c i e s (Owens, 1967; C h i n g and D o e r k s e n , 1971). T h e r e i s some d i s c r e p a n c y i n t h e l i t e r a t u r e as t o whether t h e two s m a l l p a i r s of chromsomes, 12 and 13 i n D o u g l a s - f i r a r e t e l o c e n t r i c o r s u b t e l o c e n t r i c . 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 ) , L i v i n g s t o n (1971) and Wochok e t a l . (1980) r e f e r r e d t o them as t e l o c e n t r i c w h i l e D e V e s c o v i and S z i k l a i (1975) l a b e l l e d them s u b t e l o c e n t r i c . F o r many y e a r s t h e r e were a r g u e m e n t s r a i s e d a g a i n s t t h e e x i s t e n c e o f s t a b l e t e l o c e n t r i c s i n n a t u r a l p l a n t and a n i m a l p o p u l a t i o n s . T h e o r i e s a g a i n s t t h e 71 e x i s t a n c e o f s t a b l e t e l o c e n t r i c s were a r g u e d by M c C l i n t o c k ( 1 9 3 2 ) , M u l l e r ( 1 9 4 0 ) , Rhoades ( 1 9 4 0 ) , Coleman (1943) and White ( 1 9 7 3 ) , w h i l e M akino and Momma ( 1 9 5 0 ) , Marks ( 1 9 5 7 ) , John and H e w i t t ( 1 9 6 5 ) , S t r i d ( 1 9 6 8 ) , J o hn and Freeman (1975) and J o n e s (1978) have d e m o n s t a t e d t h a t s t a b l e t e l o c e n t r i c s do e x i s t . L i v i n g s t o n (1971) t h r o u g h t h e s t u d y of m e i o s i s i n D o u g l a s - f i r , d e m o n s t r a t e d t h a t t h e c e n t r o m e r e s of chromosomes 12 and 13 were t e r m i n a l l y s i t u a t e d . The p r e s e n t s t u d y s u b s t a n t i a t e s t h e e x i s t e n c e of t h e s e two t e l o c e n t r i c chromosome p a i r s . A c o n s i s t e n t h a p l o i d chromosome number of n=12 was f o u n d i n a l l c e l l s e x a mined f o r t h e f i v e A s i a t i c s p e c i e s , PF, PS, PW, P J and PG and f o r t h e N o r t h A m e r i c a n s p e c i e s (PM), w h i c h i n c l u d e d s i x m e t a c e n t r i c and s i x s u b m e t a c e n t r i c p a i r s o f chromosomes. Th e s e o b s e r v a t i o n s a r e i n agreement w i t h t h e r e p o r t e d i n f o r m a t i o n on PM ( C h r i s t i a n s e n , 1963), PW (Thomas and C h i n g , 1968), P J , PS and PF ( D o e r k s e n and C h i n g , 1972). T h i s i s t h e f i r s t r e p o r t e d i n f o r m a t i o n on PG k a r y o t y p e a n a l y s i s w h i c h e x h i b i t s t h e same chromosome number as o b s e r v e d f o r t h e o t h e r f o u r A s i a t i c s p e c i e s . 5.2. Chromosome M o r p h o l o g y Among t h e s p e c i e s , s e v e r a l g e n e r a l t r e n d s were o b s e r v e d . T hese i n c l u d e d a d e c r e a s e i n arm l e n g t h and m o r p h o l o g i c a l i n d e x v a l u e s f o r t h e m e t a c e n t r i c s , s u b m e t a c e n t r i c s and where a p p l i c a b l e t h e t e l o c e n t r i c p a i r s i n e a c h s p e c i e s and a u n i f o r m i t y i n arm r a t i o and c e n t r o m e r e i n d e x v a l u e s f o r t h e i n d i v i d u a l m e t a c e n t r i c s and s u b m e t a c e n t r i c s w i t h i n e a c h s p e c i e s . PF was t h e o n l y s p e c i e s t h a t d e v i a t e d from t h e s e t r e n d s . The m o r p h o l o g i c a l i n d e x v a l u e s f o r chromosomes 1 t o 6 ( m e t a c e n t r i c s ) were random r a t h e r t h a n 72 d e c r e a s i n g . The s h o r t arm v a l u e f o r chromosome 2 was s m a l l e r , t h a n t h e s h o r t arm v a l u e of chromosome 3 and t h e l o n g arm v a l u e o f chromosome 4 was s m a l l e r t h a n t h e l o n g arm v a l u e of chromosome 5, d e v i a t i n g f r o m t h e o b s e r v e d t r e n d s . The arm r a t i o and c e n t r o m e r e i n d e x v a l u e s f o r chromosomes 2 and 6 were d i f f e r e n t from t h e v a l u e s of t h e o t h e r f o u r m e t a c e n t r i c p a i r s w h i c h were q u i t e u n i f o r m . T h e s e d e v i a t i o n s c a n be u s e d t o i d e n t i f y and s e p a r a t e PF f r o m t h e o t h e r s p e c i e s b e c a u s e t h e m e t a c e n t r i c s do n o t f o l l o w a c o n s i s t e n t d e c r e a s i n g t r e n d . I f i t i s assumed t h a t t h e d e c r e a s i n g t r e n d i n arm l e n g t h i s t h e norm f o r t h e P s e u d o t s u g a genus t h e n i t c a n be i m p l i e d t h a t t h e r e have been some chromosomal c h a n g e s i n PF. U n f o r t u n a t e l y , w i t h t h e i n f o r m a t i o n a v a i l a b l e i t i s not p o s s i b l e t o e l u c i d a t e t h e mode of change t h a t has o c c u r r e d i n PF. P e r h a p s w i t h f u r t h e r r e s e a r c h ( e g . chromosome b a n d i n g ) i t w i l l be p o s s i b l e t o d e t e r m i n e t h e chromosomal e v o l u t i o n o f PF. N e v e r t h e l e s s , PF can be d i f f e r e n t i a t e d from t h e o t h e r s p e c i e s i n t h e genus by t h e s t r u c t u r e and m o r p h o l o g y o f i t s m e t a c e n t r i c chromosomes. 5.3. S e c o n d a r y C o n s t r i c t i o n s The number of s e c o n d a r y c o n s t r i c t i o n s (SC) v a r i e d between s e v e r a l of t h e s p e c i e s , but g e n e r a l l y one or two c o n s i s t e n t SC were o b s e r v e d i n e a c h s p e c i e s . Two SC were o b s e r v e d on t h e l o n g arms o f chromosomes 3 and 10 f o r b o t h CDf and I D f . The p o s i t i o n s and f r e q u e n c i e s o f t h e c o n s t r i c t i o n s were s i m i l a r f o r b o t h v a r i e t i e s . The main r e a s o n t h a t SC were n o t o b s e r v e d i n 100% o f a l l c e l l s a n a l y z e d was t h a t o v e r l a p p i n g chromosomes o b s c u r e d some of t h e c o n s t r i c t i o n s . In 73 w e l l s p r e a d c e l l s t h e SC were g e n e r a l l y c l e a r l y d e f i n e d . Thomas and C h i n g (1968)''" o b s e r v e d t h r e e SC i n D o u g l a s - f i r , l o c a t e d on chromosomes 3, 10 and 11. The c o n s t r i c t i o n on chromosome 11 was o n l y o b s e r v e d 10% o f t h e t i m e so t h e y c o n c l u d e t h a t i t d i d n o t o c c u r w i t h enough r e g u l a r i t y t o be of d i a g n o s t i c u s e . The work o f Wochok e_t a l . (1980) a g r e e d w i t h t h e p r e s e n t s t u d y where two SC l o c a t e d on chromosomes 3 and 10 were o b s e r v e d . Two SC were f o u n d i n PM, l o c a t e d on t h e l o n g arms of chromosomes 2 and 4. Two SC f o r PM were p r e v i o u s l y r e p o r t e d by C h r i s t i a n s e n ( 1 9 6 3 ) . P J a l s o c o n t a i n e d two SC l o c a t e d on t h e l o n g arms o f chromosomes 2 and 4. O n l y one SC was p r e v i o u s l y r e p o r t e d f o r PJ ( D o e r k s e n and C h i n g , 1972). PF c o n t a i n e d two S C . s i m i l a r t o PM and PJ e x c e p t t h a t t h e y o c c u r r e d on chromosomes 4 and 6. One c o n s t r i c t i o n , l o c a t e d on chromosome 4 was r e p o r t e d e a r l i e r f o r PF ( D o e r k s e n and C h i n g , 1972). PG, PS and PW e a c h c o n t a i n e d one SC, l o c a t e d on chromosome 4. One SC (chromosome 4) was p r e v i o u s l y r e p o r t e d f o r PS ( D o e r k s e n and C h i n g , 1972) w h i l e t h r e e SC, l o c a t e d on chromosomes 4, 6 and 11 were r e p o r t e d f o r PW by Thomas and C h i n g ( 1 9 6 8 ) . The o b s e r v e d f r e q u e n c i e s o f o c c u r r e n c e were 100% f o r t h e SC on chromosome 4, l e s s t h a n 50% f o r t h e SC on chromosome 6 and l e s s t h a n 10% f o r t h e SC on chromosome 11. I f a l l SC t h a t o c c u r r e d a t f r e q u e n c i e s of l e s s t h a n 70% were d e l e t e d , as was p e r f o r m e d i n t h i s s t u d y , t h e n t h e r e s u l t s o f Thomas and C h i n g (1968) would a g r e e w i t h t h e r e s u l t s p r e s e n t e d i n t h i s work, s i n c e o n l y one SC (chromosome 4) was o b s e r v e d a t a f r e q u e n c y above 70%. 74 From t h e i d i o g r a m s i n F i g u r e 10, s e v e r a l g e n e r a l i z a t i o n s c a n be made. A l l of t h e n=12 s p e c i e s c o n t a i n a SC on chromosome 4. The s e s p e c i e s c an be d i v i d e d i n t o t h r e e g r o u p s b a s e d on number and p o s i t i o n o f SC. PG, PS and PW e a c h c o n t a i n one SC, P J and PM c o n t a i n a SC on chromosomes 2 and 4, w h i l e PF c o n t a i n s a SC on chromosomes 4 and 6. SC c o u l d p o s s i b l y be u s e d t o i d e n t i f y PF, but i t ' s n o t p o s s i b l e t o i d e n t i f y any o f t h e o t h e r n=12 s p e c i e s . B o t h v a r i e t i e s of D o u g l a s - f i r c o n t a i n SC on d i f f e r e n t chromosomes (3 and 10) t h a n t h e n=12 s p e c i e s (2, 4 o r 6 ) . An i n t e r e s t i n g o b s e r v a t i o n i s t h a t t h e most c o n s i s t e n t c o n s t r i c t i o n i n t h e n=l2 s p e c i e s i s l o c a t e d on chromosome 4, w h i l e i n D o u g l a s -f i r i t s f o u n d on chromosome 3. S e v e r a l r e s e a r c h e r s have p u t f o r t h t h a t a s i m p l e e x p l a n a t i o n f o r t h e e x t r a chromosome p a i r i n D o u g l a s - f i r i s t h a t one of t h e m e t a c e n t r i c s s p l i t a t t h e c e n t r m e r e t o form two chromosomes w i t h t e r m i n a l c e n t r o m e r e s (Thomas and C h i n g , 1968; D o e r k s e n and C h i n g , 1972). S u p p o s i n g t h a t D o u g l a s - f i r d i d a r i s e from t h e f i s s i o n o f one o f t h e f i r s t t h r e e m e t a c e n t r i c s of an n=l2 a n c e s t o r , t h e chromosome w i t h t h e SC • would s h i f t from p o s i t i o n 4 t o p o s i t i o n 3 i n r e l a t i v e l e n g t h . The new s p e c i e s would l a c k one m e t a c e n t r i c p a i r and have an e x t r a two t e l o c e n t r i c p a i r s r e l a t i v e t o t h e o r i g i n a l n=12 s p e c i e s . F u r t h e r r e s e a r c h i s r e q u i r e d t o d e t e r m i n e i f t h i s h y p o t h e t i c a l e l u c i d a t i o n o f t h e e v o l u t i o n of D o u g l a s - f i r from an n=l2 a n c e s t o r i s r e m o t e l y c o r r e c t . The s h i f t i n t h e p o s i t i o n o f t h e most c o n s i s t e n t SC t e n d s t o s u p p o r t t h e h y p o t h e s i s , b u t w i t h o u t f u r t h e r i n v e s t i g a t i o n u s i n g chromosome b a n d i n g o r gene mapping no c o n c l u s i o n s c a n be made c o n c e r n i n g t h e e v o l u t i o n o f D o u g l a s - f i r r e l a t i v e t o t h e o t h e r P s e u d o t s u g a s p e c i e s . 75 5.4. C o a s t a l V e r s u s I n t e r i o r D o u q l a s - f i r (n=13) The r e s u l t s o f t h e t - t e s t ( T a b l e 11) c o m p a r i n g 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 d i s p l a y h i g h l y s i g n i f i c a n t d i f f e r e n c e s ( p <0.0l) f o r t h e v a l u e s MI 1, 2, 3, 4, 8, 9, 10 and 11 and RL 1, 3, 6, 10, 12 and 13. The g r e a t e s t v a r i a t i o n was f o u n d between RL6 ( t = 6.57), RL13 ( t = 5.64), RL 1 ( t = 3 . 4 l ) and RL12 ( t = 3.34). T h e s e o b s e r v a t i o n s c o i n c i d e w i t h numerous e a r l i e r o b s e r v a t i o n s , on m o r p h o l o g i c a l , p h y s i o l o g i c a l and n u c l e a r d i f f e r e n c e s between c o a s t a l and i n t e r i o r p r o v e n a n c e s . C l e a r m o r p h o l o g i c a l d i f f e r e n c e s between c o a s t a l and i n t e r i o r p r o v e n a n c e s have been d e s c r i b e d f o r s e e d c h a r a c t e r i s t i c s ( A l l e n , 1960; R o b i n s o n , 1963; D u n l a p , 1964; S z i k l a i , 1969), s c a l e and b r a c t c h a r a c t e r i s t i c s (Yao, 1971), n e e d l e t h i c k n e s s , cone d i a m e t e r and cone s p e c i f i c g r a v i t y ( T u s k o , 1963), r a t e of g e r m i n a t i o n ( A l l e n and B i e n t j e s , 1954; A l l e n , 1961), n u c l e a r volume ( E l - L a k a n y and S z i k l a i , 1973), t e r p e n e a n a l y s i s (Hanover and F u r n i s s , 1966; Von R u d l o f f , 1972) and a l l o z y m e f r e q u e n c i e s (Yeh and O ' M a l l e y , 1978). The o n l y o t h e r s t u d y t h a t compared t h e k a r y o t y p 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 , D e V e s c o v i and S z i k l a i ( 1 9 7 5 ) , d i d n o t f i n d any v a r i a t i o n between the RL of t h e two v a r i e t i e s but t h e y d i d f i n d some v a r i a t i o n i n chromosome volume. T h e i r s t u d y i n c l u d e d t h r e e c o a s t a l (two i n C a l i f o r n i a , one i n W a s h i n g t o n ) and one i n t e r i o r p r o v e n a n c e ( W a s h i n g t o n ) , whereas t h i s s t u d y i n c l u d e d s e v e n c o a s t a l and s i x i n t e r i o r p r o v e n a n c e s e x t e n d i n g from c e n t r a l B.C. t o n o r t h e r n C a l i f o r n i a . The l a c k of v a r i a t i o n i n chromosome c h a r a c t e r i s t i c s may have been due t o a s m a l l sample s i z e , c o n f i n e d t o a narrow r e g i o n of t h e D o u g l a s - f i r r a n g e . The c o a s t a l 76 p r o v e n a n c e l o c a t e d i n W a s h i n g t o n was f o u n d t o c o n t a i n 1.5 t i m e s more chromosome volume t h a n t h e i n t e r i o r p r o v e n a n c e , s u b s t a n t i a t i n g p r e v i o u s work a s f a r as v a r i a t i o n i n n u c l e a r volume and DNA c o n t e n t s a r e c o n c e r n e d . S i n c e CDf and IDf have d i v e r g e d t o t h e p o i n t t h a t m o r p h o l o g i c a l and p h y s i o l o g i c a l d i f f e r e n c e s a r e n o t i c e a b l e between t h e two v a r i e t i e s , i t i s n o t u n e x p e c t e d t h a t s u b t l e v a r i a t i o n s i n chromosomal c h a r a c t e r i s t i c s have a l s o t a k e n p l a c e . The m u l t i v a r i a t e a n a l y s i s f u r t h e r c o n f i r m s t h e r e s u l t s t h a t s u b t l e c h a n g e s t o t h e k a r y o t y p e s of 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 o c c u r r e d . U s i n g o n l y 8% o f t h e i n p u t v a r i a b l e s , t h e d i s c r i m i n a n t a n a l y s i s was a b l e t o s e p a r a t e t h e two D o u g l a s - f i r v a r i e t i e s a t t h e 1% l e v e l . The j a c k - k n i f e d c l a s s i f i c a t i o n p r o c e d u r e y i e l d e d an 86.2% c o r r e c t c l a s s i f i c a t i o n f o r t h e i n d i v i d u a l k a r y o t y p e s o f e a c h sample. The o v e r l a p r e g i o n d e p i c t e d i n t h e h i s t o g r a m ( F i g u r e 11) s u g g e s t s t h a t 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 a r e not c o m p l e t e l y i s o l a t e d . These r e s u l t s a g r e e w i t h numerous e a r l i e r o b s e r v a t i o n s on m o r p h o l o g i c a l , p h y s i o l o g i c a l and n u c l e a r d i f f e r e n c e s between CDf and I D f . W h i l e d i f f e r e n c e s were f o u n d between c o a s t and i n t e r i o r p r o v e n a n c e s t h e r e were d e f i n i t e o v e r l a p s i n t h e p r o v e n a n c e s b o r d e r i n g t h e t r a n s i t i o n z o n e . T h e s e were f o u n d f o r s e e d c h a r a c t e r i s t i c s ( A l l e n , 1960; R o b i n s o n , 1963; D u n l a p , 1964; S z i k l a i , 1969), cone s c a l e and b r a c t c h a r a c t e r i s t i c s (Yao, 1971), and n u c l e a r volume ( E l - L a k a n y and S z i k l a i , 1971; 1973). No b a r r i e r s t o r a c i a l c r o s s e s o f D o u g l a s - f i r have been r e p o r t e d ( S i l e n , 1978). T h i s i m p l i e s t h a t even t h o u g h c o a s t and i n t e r i o r D o u g l a s - f i r have d i v e r g e d , t h e v a r i a t i o n . i s not enough 77 t o p r e v e n t h y b r i d i z a t i o n between t h e two v a r i e t i e s . 5.5. V a r i a t i o n Among The N=12 Spec i e s From t h e Duncan's M u l t i p l e Range T e s t i t was n o t e d t h a t s e v e r a l 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 were s i g n i f i c a n t l y d i f f e r e n t (p<0.05) f o r one o r more s p e c i e s when compared t o t h e r e s t . U t i l i z i n g t h i s i n f o r m a t i o n , i t i s p o s s i b l e t o d e v i s e a s y s t e m u s i n g t h e chromosomal c h a r a c t e r i s t i c s t o s e p a r a t e and i d e n t i f y t h e i n d i v i d u a l s p e c i e s . PF: t h e AR v a l u e s ( T a b l e 15) of PF f o r chromosomes 2 ( 1 . 2 5 ) , 5 ( 1 . 1 3 ) , 7 (2.45) and 8 (2.49) a r e s i g n i f i c a n t l y d i f f e r e n t (p<0.05) from t h e o t h e r s p e c i e s , t h e r e f o r e , t h e s e v a l u e s c o u l d be us e d t o s e p a r a t e and i d e n t i f y PF from t h e r e m a i n i n g s p e c i e s . S i n c e AR and CI a r e c l o s e l y r e l a t e d , t h e CI v a l u e s f o r chromosomes 2, 5, 7 and 8 a r e a l s o s i g n i f i c a n t l y d i f f e r e n t (p<0.05) f o r PF ( T a b l e 1 6 ) . The MI v a l u e of chromosome 2 (54.70) i s u n i q u e t o PF ( T a b l e 1 7 ) . The RL v a l u e s of chromosomes 3 (123) and 10 (77) a r e a l s o s i g n i f i c a n t l y d i f f e r e n t (p<0.05) from t h e o t h e r s p e c i e s ( T a b l e 1 8 ) . A l l of t h e s e c h a r a c t e r i s t i c s c an be u s e d t o s e p a r a t e and i d e n t i f y PF from t h e o t h e r members o f t h e P s e u d o t s u g a genus. PW: t h e AR v a l u e s o f PW ( T a b l e 15) f o r chromosomes 2 (1.10) and 8 (2.37) as well--as t h e CI v a l u e s ( T a b l e 16) f o r chromosomes 2 (47.71) and 8 (29.70) d i f f e r s i g n i f i c a n t l y (p<0.05) from t h e o t h e r s p e c i e s . The RL o f chromosome 1 (137) i s u n i q u e t o t h i s s p e c i e s ( T a b l e 1 8 ) . U t i l i z i n g t h e s e f i v e c h a r a c t e r i s t i c s i t i s p o s s i b l e t o s e p a r a t e and i d e n t i f y t h i s s p e c i e s . PM: t h e RL of a l l t h e PM chromosomes e x c e p t chromosome 6 78 ( T a b l e 18) a r e s i g n i f i c a n t l y d i f f e r e n t (p<0.05) from t h e A s i a t i c s p e c i e s , t h e r e f o r e b a s e d o n l y on RL, PM can be d i s t i n g u i s h e d from t h e o t h e r s p e c i e s i n t h e P s e u d o t s u g a g e n u s . PG: t h e AR v a l u e s o f chromosomes 2 ( 1 . 0 4 ) , 9 (2.27) and 10 (2.28) ( T a b l e 15) as w e l l as t h e CI v a l u e s ( T a b l e 16) f o r chromosomes 2 ( 4 8 . 9 6 ) , 9 (30.58) and 10 (30.55) a r e s i g n i f i c a n t l y d i f f e r e n t (p<0.05) f o r PG, so t h e s e c h a r a c t e r i s t i c s c a n be u s e d t o s e p a r a t e and i d e n t i f y t h i s s p e c i e s . PS: t h e r e i s not one chromosomal c h a r a c t e r i s t i c of PS t h a t d i f f e r s s i g n i f i c a n t l y f r o m a l l o t h e r s p e c i e s . I t i s s t i l l p o s s i b l e t o i d e n t i f y PS by u t i l i z i n g more t h a n one c h a r a c t e r i s t i c and a p p l y i n g t h e p r o c e s s o f e l i m i n a t i o n . The AR v a l u e s f o r chromosomes 7 t o 12 ( T a b l e 15) a s s i g n s PS and PM t o t h e same s i g n i f i c a n c e g r o u p . I t was p r e v i o u s l y n o t e d t h a t PM has v e r y d i s t i n c t i v e RL v a l u e s ( T a b l e 18), so b y ' t h e p r o c e s s of e l i m i n a t i o n PS c a n be d i s t i n g u i s h e d f r o m PM by RL v a l u e s . P J : as was o b s e r v e d w i t h PS, t h e r e a r e no chromosomal c h a r a c t e r i s t i c s o f P J t h a t a r e s i g n i f i c a n t l y d i f f e r e n t f r o m a l l o t h e r s p e c i e s . However, u s i n g t h e AR v a l u e s and t h e p r o c e s s o f e l i m i n a t i o n i t i s p o s s i b l e t o i d e n t i f y t h i s s p e c i e s . From e a r l i e r o b s e r v a t i o n s i t was f o u n d t h a t t h e most d i s c r i m i n a t i n g c h a r a c t e r i s t i c was t h e AR v a l u e of chromosome 2 ( T a b l e 15), where i t i s p o s s i b l e t o i d e n t i f y t h r e e s p e c i e s , PG, PW and PF. PS, PM and PJ a r e n o t s i g n i f i c a n t l y d i f f e r e n t (p<0.05), hence a r e a s s i g n e d t o t h e same g r o u p . I f an unknown has an AR v a l u e t h a t b e l o n g s t o t h i s g r o u p , i t can be assumed t o be one of t h e s e t h r e e s p e c i e s . The AR v a l u e s of chromosomes 7 t o 12 a s s i g n s PS and PM t o one c l a s s ( g r o u p one) and PJ t o a n o t h e r s i g n i f i c a n t l y 79 d i f f e r e n t g r o u p , so by t h e p r o c e s s of e l i m i n a t i o n P J can be s e p a r a t e d from PM and PS. T h i s i s not t h e o n l y method t o d e t e r m i n e i f an unknown i s P J , b ut i t i s t h e most s t r a i g h t f o r w a r d and l e a s t c o n f u s i n g of a l l p o s s i b l e methods. T h e r e f o r e , b a s e d on chromosomal c h a r a c t e r i s t i c s , i t i s p o s s i b l e t o s e p a r a t e and i d e n t i f y t h e s i x n=12 s p e c i e s i n t h e P s e u d o s t s u g a g e n u s . The m u l t i v a r i a t e a n a l y s i s c o n f i r m s t h e a n a l y s i s of v a r i a n c e r e s u l t s t h a t t h e s i x n=l2 s p e c i e s can be s e p a r a t e d and i d e n t i f i e d by chromosomal v a r i a t i o n . O n l y 17% of t h e v a r i a b l e s were r e q u i r e d t o d i s c r i m i n a t e among t h e f i v e A s i a t i c s p e c i e s and PM. PM was r e a d i l y d i s t i n g u i s h a b l e f r o m t h e A s i a t i c s p e c i e s by t h e f i r s t c a n o n i c a l v a r i a b l e i m p l y i n g t h a t PM has undergone a h i g h e r d e g r e e of chromosomal d i v e r g e n c e f r o m t h e A s i a t i c s p e c i e s compared t o t h e chromosomal change w i t h i n t h e A s i a t i c s p e c i e s . When t h e f i v e A s i a t i c s p e c i e s were a n a l y z e d s e p a r a t e l y , a l l s p e c i e s were s i g n i f i c a n t l y d i f f e r e n t a t t h e 1% l e v e l i m p l y i n g t h a t a m e a s u r e a b l e amount of chromosome change has o c c u r r e d w i t h i n t h e A s i a t i c s p e c i e s a s w e l l as between t h e A s i a t i c and N o r t h A m e r i c a n spec i e s . The m u l t i v a r i a t e a n a l y s i s i s a p o w e r f u l t o o l f o r u n r a v e l l i n g complex r e l a t i o n s h i p s between v a r i a b l e s . T h i s t e c h n i q u e a l l o w s d i s c r i m i n a t i o n of known s p e c i e s i n t h r e e d i m e n s i o n s and a l s o can be expanded t o c l a s s i f y "unknown" samples as t o s p e c i e s o r i g i n . The d i s c r i m i n a t i o n was v e r y good f o r t h e s p e c i e s u s e d i n s p i t e of the s i m i l a r i t y o f n u m e r i c a l d a t a among t h e d i f f e r e n t s p e c i e s . 80 C h a r a c t e r i z a t i o n of t h e d i f f e r e n t s p e c i e s c o u l d be a c h i e v e d w i t h a h i g h d e g r e e of r e l i a b i l i t y w i t h t h e use o f t h i s s t a t i s t i c a l t e c h n i q u e . The i n f o r m a t i o n p r e s e n t e d s t r o n g l y i n d i c a t e s t h a t t h e b a s i c chromosome number i n t h e genus P s e u d o t s u g a i s 12, and t h a t D o u g l a s - f i r w i t h an n-number of 13 a r o s e f r o m a s p e c i e s w i t h an n-number of 12. PM was h y b r i d i z e d w i t h D o u g l a s - f i r and p r o d u c e d a few v i a b l e p r o g e n y when PM was t h e p o l l e n p a r e n t ( C h i n g , 1959). No s u c c e s s f u l c r o s s has been made between D o u g l a s - f i r and t h e A s i a t i c s p e c i e s d e s p i t e many t r i a l s , p a r t i c u l a r l y by O r r - E w i n g o f t h e B r i t i s h C o l u m b i a F o r e s t S e r v i c e ( O r r - E w i n g e t a l . 1972). A l t h o u g h d i f f e r e n c e s i n chromosome number i s s u s p e c t i n t h e l a c k of s u c c e s s , t h i s does not a p p l y t o c r o s s e s u s i n g PM as p o l l e n . O t h e r c a u s e s may be i n v o l v e d a s s e c t i o n e d m i c r o p y l a r c a n a l s r e v e a l e d t h a t PW p o l l e n expanded l o n g i t u d i n a l l y o n l y a b o u t 200 m i c r o n s , a d i s t a n c e i n s u f f i c i e n t t o c o n t a c t t h e n u c e l l a r t i s s u e ( O r r - E w i n g e t a l . 1972; S i l e n , 1978). B a s e d on t h e v a r i a t i o n s between s p e c i e s a l l members o f t h e P s e u d o t s u g a genus have d i v e r g e d t o v a r y i n g d e g r e e s , w i t h t h e most o b v i o u s change o c c u r r i n g t o D o u g l a s - f i r . 81 6. SUMMARY A c o m p a r a t i v e k a r y o t y p e s t u d y o f se v e n s p e c i e s i n t h e genus P s e u d o t s u g a was c o n d u c t e d . T h e s e i n c l u d e d two N o r t h A m e r i c a n s p e c i e s , m a c r o c a r p a and P_;_ m e n z i e s i i , t h e l a t t e r c o n s i s t i n g of two v a r i e t i e s , P_^  m e n z i e s i i v a r . menz i e s i i ( c o a s t a l D o u g l a s - f i r ) and P_j_ m e n z i e s i i v a r . g l a u c a , ( i n t e r i o r D o u g l a s - f i r ) and f i v e A s i a t i c s p e c i e s , P_^  f o r r e s t i i , P. s i n e n s i s , P. g a u s s e n i i , P. w i l s o n i a n a and P_;_ j a p o n i c a . The s p e c i e s were compared u s i n g chromosome number, s e c o n d a r y c o n s t r i c t i o n s and chromosomal c h a r a c t e r i s t i c s i n c l u d i n g arm r a t i o , c e n t r o m e r e i n d e x , m o r p h o l o g c i a l i n d e x and r e l a t i v e l e n g t h . (1) Chromosome Number A s o m a t i c chromosome number-of 2n=26 was o b s e r v e d f o r t h e two v a r i e t i e s o f D o u g l a s - f i r , w h i l e a s o m a t i c chromosome number of 2n = 24 was o b s e r v e d f o r Pj_ m a c r o c a r p a and t h e f i v e A s i a t i c s p e c i e s s t u d i e d . T h i s was t h e f i r s t known r e p o r t e d k a r y o t y p e anaJ L y s i s o f P^ g a u s s e n i i , w h i c h f o l l o w e d t h e t r e n d o f t h e o t h e r A s i a t i c s p e c i e s . D o u g l a s - f i r chromosome p a i r s were c l a s s i f i e d i n t o f i v e m e t a c e n t r i c s , s i x s u b m e t a c e n t r i c s and two t e l o c e n t r i c s . The n=12 s p e c i e s c o n t a i n e d s i x m e t a c e n t r i c s and s i x s u b m e t a c e n t r i c s . D o u g l a s - f i r c o u l d be s e p a r a t e d from t h e r e m a i n i n g s p e c i e s b a s e d on chromosome number a l o n e . 82 (2) S e c o n d a r y C o n s t r i c t i o n s The number and f r e q u e n c y of s e c o n d a r y c o n s t r i c t i o n s v a r i e d w i t h i n s p e c i e s , w i t h s e v e r a l c o n s t r i c t i o n s o c c u r r i n g a t f r e q u e n c i e s above 70%. (3) C o a s t a l v e r s u s I n t e r i o r D o u g l a s - f i r 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 c o u l d be d i f f e r e n t i a t e d u s i n g s e v e r a l chromosomal c h a r a c t e r i s t i c s ; w i t h t h e r e l a t i v e l e n g t h of chromosome 6 b e i n g t h e most d i s c r i m i n a t i n g v a r i a b l e . A h i g h l e v e l of d i s c r i m i n a t i o n was o b s e r v e d between t h e two D o u g l a s - f i r v a r i e t i e s when t h e d i s c r i m i n a n t f u n c t i o n a n a l y s i s was u s e d . A h i s t o g r a m o f t h e a c t u a l sample p o i n t s d i s p l a y e d a s m a l l o v e r l a p r e g i o n between 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 . (4) V a r i a t i o n Between The N=12 S p e c i e s U t i l i z i n g s e v e r a l chromosomal c h a r a c t e r i s t i c s and t h e p r o c e s s of e l i m i n a t i o n , a scheme was d e v i s e d t o i d e n t i f y and s e p a r a t e t h e s i x n=l2 s p e c i e s . The arm r a t i o o f chromosome 2 i d e n t i f i e d P_;_ f o r r e s t i i , P. g a u s s e n i i and P^ w i l s o n i a n a . U s i n g t h e arm r a t i o v a l u e s of chromosomes 7 t o 12, P_;_ j a p o n i c a c o u l d be i d e n t i f i e d w h i l e t h e r e l a t i v e l e n g t h s of chromosomes 1 t o 5 o r 7 t o 12 would s e p a r a t e and i d e n t i f y P_;_ m a c r o c a r p a from P^ s i n e n s i s . Good d i s c r i m i n a t i o n was shown among P_^_ m a c r o c a r p a and t h e A s i a t i c s p e c i e s when t h e d i s c r i m i n a n t f u n c t i o n a n a l y s i s was u s e d . The d i s c r i m i n a t i o n among t h e A s i a t i c s p e c i e s was f u r t h e r i m p r o v e d when t h e w e l l s e p a r a t e d P_^_ m a c r o c a r p a was o m i t t e d . T h i s s t a t i s t i c a l t e c h n i q u e was p a r t i c u l a r l y h e l p f u l i n s e l e c t i n g v a r i a b l e s t h a t b e s t s e p a r a t e d t h e t a x a . F i n a l l y , t h e s t u d y has shown t h a t s e v e n s p e c i e s of t h e genus P s e u d o t s u g a can be i d e n t i f i e d and s e p a r a t e d by chromosomal 83 m o r p h o l o g y . The r e s u l t s s t r o n g l y s u g g e s t t h a t D o u g l a s - f i r a r o s e f r o m a s p e c i e s w i t h an n-number o f 12. 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 c a n a l s o be s e p a r a t e d and i d e n t i f i e d u s i n g chromosomal c h a r a c t e r i s t i c s . 84 7. REFERENCES A l l e n , G.S. and W. B i e n t j e s . 1954. S t u d i e s on c o n i f e r o u s t r e e s e e d a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a . F o r . C h r o n . 30:184-196. A l l e n , G.S. 1960. A method of d i s t i n g u i s h i n g c o a s t a l f r o m i n t e r i o r D o u g l a s - f i r . B r i t i s h C o l u m b i a Lumberman, 44:26-30. A l l e n , G.S. 1961. T e s t i n g of D o u g l a s - f i r s e e d f o r p r o v e n a n c e . P r o c . I n t . Seed T e s t . A s s n . 26:388-403. B a r n e r , H. and H.. C h r i s t i a n s e n . 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 of t h e most f a v o u 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 m e n z i e s i i , S i l v a e G e n e t . 11:89-102. Bean, W.J. 1976. T r e e s and s h r u b s h a r d y i n t h e B r i t i s h I s l e s . V o l . 3. N-Rh. J o h n M u r r a y L t d . , L o n d o n . Chengde, C. 1981. A b r i e f i n t r o d u c t i o n t o t h e C h i n e s e S p e c i e s o f t h e Genus PSEUDOTSUGA. D a v i d s o n i a , 12:15-17. C h i n g , K.K. 1959. H y b r i d i z a t i o n between 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 . F o r . S c i . 5:246-254. C h i n g , K.K. and A. D o e r k s e n , 1971. A n a t u r a l c h i m e r a of D o u g l a s - f i r . S i l v a e G e n e t . 20:209-210. C h r i s t i a n s e n , H. 1972. On t h e d e v e l o p m e n t of p o l l e n and t h e f e r t i l i z a t i o n mechanism of L a r i x and P s e u d o t s u g a  m e n z i e s i i . S i l v a e G e n e t . 21:166-174. C h r i s t i a n s e n , H. 1963. On t h e chromosomes of P s e u d o t s u g a -macrocarpa and P s e u d o t s u g a menz i e s i i . S i l v a e G e n e t . 12: 124-127. Coleman, L.C. 1943. Chromosome s t r u c t u r e i n t h e A c r i d i d e a w i t h s p e c i a l r e f e r e n c e t o t h e X-chromosome. G e n e t i c s , • 28:2-8. D a l l i m o r e , W. and A.B. J a c k s o n . 1966. A handbook o f C o n i f e r a e and G i n g k o a c e a e . Edward A r n o l d , L o ndon. D e V e s c o v i , M.A. and 0. S z i k l a i . 1975. C o m p a r a t i v e k a r y o t y p e a n a l y s i s o f D o u g l a s - f i r . S i l v a e G e n e t . 24:68-72. D o e r k s e n , A.H. and K.K. C h i n g . 1972. K a r y o t y p e i n t h e genus P s e u d o t s u g a . F o r . S c i . 18:66-69. 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J o h n , B. and G.M. H e w i t t . 1965. K a r y o t y p e s t a b i l i t y and DNA v a r i a b i l i t y i n t h e A c r i d i d a e . Chromosoma, 16:584-578. J o h n , B. and M. Freeman. 1975. C a u s e s and c o n s e q u e n c e s of R o b e r t s o n i a n e x c h a n g e . Chromosoma, 52:123-136. J o n e s , K. 1978. A s p e c t s of chromosomal e v o l u t i o n i n h i g h e r p l a n t s . Adv. B o t . Res. 6:120-194. L a n g l e t , 0. 1934. S v e n s k a s k o g s v a r d s f o r e n . T i d s k r . 32:1-11. L e v a n , A., K. F r e d g a and A.A. S a n d b e r g . 1964. N o m e n c l a t u r e f o r t h e c e n t r o m e r i c p o s i t i o n on chromosomes. H e r e d i t a s , 52:201-220. L i v i n g s t o n , G.K. 1971. The m o r p h o l o g y and b e h a v i o u r o f m e i o t i c chromosomes o f D o u g l a s - f i r . S i l v a e . G e n e t . 20:75-82. L i t t l e , E.R. 1952. The genus P s e u d o t s u g a ( D o u g l a s - f i r ) i n N o r t h A m e r i c a . L e a f t l . West. B o t . A p r i l 181-196. Makino, S. and E. Momma. 1950. O b s e r v a t i o n on t h e s t r u c t u r e o f g r a s s h o p p e r chromosomes s u b j e c t e d t o a new a c e t o c a r m i n e t r e a t m e n t . J . of Morph. 86:229-252. Marks, G.E. 1957. T e l o c e n t r i c chromosomes. Am. N a t . 91:223-232. M c C l i n t o c k , B. 1932. A c o r r e l a t i o n of r i n g - s h a p e d chromosomes w i t h v a r i e g a t i o n i n Zea mays. P r o c . N a t . Ac a d . S c i . 18:677-681. M u l l e r , H.J. 1940. An a n a l y s i s o f t h e p r o c e s s o f s t r u c t u r a l change i n t h e chromosomes o f D r o s o p h i l a . J . G e n e t . 40:1-16. O r r - E w i n g , A.L., A.R. F r a s e r and I . K a r l s s o n . 1972. I n t e r r a c i a l c r o s s e s w i t h D o u g l a s - f i r ; e a r l y f i e l d r e s u l t s . B.C. F o r . S e r v . R es. Note 55, 33 pp. Owens,- J.N. 1967. Chromosome a b e r r a t i o n s i n D o u g l a s - f i r . 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