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Three western species of the genus Habenaria willd : their relationship and crossability Fisher, Emmy H. 1974

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THREE WESTERN SPECIES OF THE GENUS H/BENARIA WILLD. THEIR RELATIONSHIP AND CROSSABILITY. 3Y EMMY H. FISHER B. Com., U n i v e r s i t y of Economics Vienna ( A u s t r i a ) , 1922 THIS THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Botany We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA. In presenting t h i s thes is in p a r t i a l f u l f i l m e n t o f the requirements f> an advanced degree at the Univers i ty of B r i t i s h Columbia, I aqree that the L ibrary shal l make it f r e e l y a v a i l a b l e for reference and s t u d / . I further agree that permission for extensive copying of th is t h e s i s for s c h o l a r l y purposes may be granted by the Head of my Department r>r by his representat ives . It is understood that copying or p u b l i c a t i o n of th is thesis f o r f i n a n c i a l gain sha l l not be allowed without my written permission. Depa rtment The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada A B S T R A C T . R e l a t i o n s h i p and i n t e r f e r t i l i t y o f H a b e n a r i a d i l a t a t a ( l u r s h ) Hook.,H. hyperborea ( L . ) R.Br, and H. s a c c a t a Greene was s t u d i e d . I n t r a s p e c i f i c and i n t e r s p e c i f i c c r o s s e s were made. Chromosome counts of the t h r e e s p e c i e s showed 21 p a i r s o f chromosomes i n the c e l l s , except f o r a s m a l l g r e e n - f l o w e r e d p o p u l a t i o n from Manning P a r k w i t h n = 42 which was c o n s i d e r e d t e t r a p l o i d and p o s s i b l y of h y b r i d o r i g i n . These counts agree w i t h e a r l i e r ones f o r the t h r e e s p e c i e s . S i n c e c r e a t i o n of the genus H a b e n a r i a Willd» these s p e c i e s have been i n c l u d e d under t r i b e Ophrydeae, which now has been changed t o O r c h i d e a e , s u b t r i b e O r c h i d i n a e t o conform w i t h the r u l i n g s of the I n t e r n a t i o n a l Code o f B o t a n i c a l Nomenclature (1959)* I n s p i t e of a p p a r e n t l y c l o s e r e l a t i o n -s h i p s the s p e c i e s m a i n t a i n t h e i r d i s t i n c t n e s s , even when growing sym-p a t r i c a l l y , i n d i c a t i n g b a r r i e r s to o u t c r o s s i n g , o r f o r p l a n t s growing i n n o r t h e r l y r e g i o n s , a l a c k of p o l l i n a t o r s . Autogamous t e n d e n c i e s have t h e r e f o r e d eveloped and H. d i l a t a t a and H. hyperborea are o u t c r o s s i n g or autogamous when the need a r i s e s . H. s a c c a t a seems to be s e l f - s t e r i l e . M i c r o s p o r o g e n e s i s i n the s p e c i e s s t u d i e d f o l l o w s t h a t of o t h e r o r c h i d s . The a r c h e s p o r i a l c e l l d i r e c t l y becomes the spore mother c e l l . A l l descendants of t h i s c e l l s t a y t o g e t h e r and d i v i d e t o g e t h e r , f o r m i n g a massula or p o l l e n p a c k e t . P o l l e n m i t o s i s r e s u l t s i n the 2 - n u c l e a t e p o l l e n g r a i n which i s shed as s u c h . The g e n e r a t i v e n u c l e u s d i v i d e s i n the p o l l e n tube, p r o d u c i n g the 2 sperm n u c l e i , as the tube e n t e r s the o v a r y . Only the c h a l a z a l megaspore i s f u n c t i o n a l . Three s i m u l t a n e o u s d i v i s i o n s produce the monosporic, 8 - n u c l e a t e Polygonum-type embryo s a c . F u s i o n o f the p o l a r n u c l e i i s the r u l e and t a k e s p l a c e b e f o r e f e r t i l i z a t i o n . T r i p l e f u s i o n f o l l o w s , hut the p r i m a r y endosperm n u c l e u s b e g i n s to degenerate u s u a l l y b e f o r e the r-ypote s t a r t s to d i v i d e . An h a u s t o r i a l susy ensor d e v e l o p s , which does not take p a r t i n the c o n s t r u c t i o n of the embryo p r o p e r . The mature embryo i s an un d i f f e r e n t i a t e d body of 50-60 c e l l s , suspended i n the a i r - f i l l e d c a -v i t y of the r e t i c u l a t e t e s t a . It t a k e s from 3-4 weeks from p o l l i -n a t i o n t o s a t u r a t i o n o f the embryo. I n t r a s p e c i f i c c r o s s e s were a l l s u c c e s s f u l . I n t e r s p e c i f i c c r o s -ses produced a h i g h e r percentage of seed w i t h w e l l d e v e l o p e d embryos i n IT. hyperborea x H. s a c c a t a c r o s s e s than i n c r o s s e s between H. riil t a t a and e i t h e r o f the 2 other s p e c i e s . The t e t r a p l o i d p l a n t s were s u c c e s s f u l both as seed and p o l l e n p a r e n t s . Regular m e i o s i s would i n d i c a t e a l l o p o l y p l o i d o r i g i n . A r t i f i c i a l p o l l i n a t i o n s showed t h a t gene f l o w i s p o s s i b l e and t h a t a r t i f i c i a l c r o s s e s are easy to make. In n a t u r e i s o l a t i n g mec'r an isms must r r e v e n t the s p e c i e s from l o s i n g t h e i r i d e n t i t y a l -though h y b r i d i z a t i o n may take p l a c e under f a v o u r a b l e c o n d i t i o n s . C o n t r o l p l a n t s of H. d i l a t a t a and H, hyperhorea not emasculated and not p r o t e c t e d showed a f u l l seed s e t , i n d i c a t i n g autocamy, whereas u n p o l l i n a t e d H. s a n c a t a y i e l d e d o n l y empty s e e d . TABLE OF CONTENTS. Page ABSTRACT i i - i i i LIST OF TABLES v LIST OF FIGURES v i ACKNOWLEDGEMENTS v i i HABENARIA DILATATA (PURSH) HOOKER).. v i i INTRODUCTION 1 -6 MATERIALS AND METHODS 7 - 1 2 O B S E R V A T I O N S AND D I S . C U S S I O N S ik-I . HABENARIA MORPHOLOGY 1 4 - 1 8 I I . MORPHOLOGY OF H. DILATATA, H. HYPERBOREA AND H. SACCATA 1 9 - 2 3 I I I . MICROSPOROGENESIS AND THE MALE GAMETOPHYTE 2 ^ - 3 0 IV. MEGASPOROGENESIS AND THE FEMALE GAMETOPHYTE 3 1 - 5 3 V. CHROMOSOME COUNTS 5 ^ - 5 6 Problems encountered 57 Autogamy v e r s u s o u t c r o s s i n g 5 8 - 5 9 D i p l o i d y v e r s u s p o l y p l o i d y 6 0 - 6 3 T a b l e s V.-IX 6 4 - 6 9 V I . GENERAL CONCLUSIONS 7 0 - 7 1 V I I . SUMMARY 72 V I I . BIBLIOGRAPHY 7 3 - 7 6 LIST OF TABLES. Table Page I . C l a s s i f i c a t i o n o f H. d i l a t a t a , H. hyperborea and H. s a c c a t a 5 - 6 I I . C o l l e c t i o n l o c a t i o n s o f the t h r e e s p e c i e s 10 I I I . F l o r e t morphology 22 IV. Stages i n o v u l e development at g i v e n times from p o l l i n a t i o n t o m a t u r i t y . 40 V. Chromosome counts 64 V I . Mean v a l u e o f f e r t i l i t y i n c r o s s e s made 66 V I I . C r o s s e s w i t h H. d i l a t a t a as seed p a r e n t 67 V I I I . C r o s s e s w i t h H. hyperborea as seed p a r e n t 68 IX. C r o s s e s w i t h H. s a c c a t a as seed p a r e n t 69 LIST OF FIGURES. F i g u r e Pa 1 Map of c o l l e c t i o n l o c a t i o n s i n B r i t i s h Columbia . . . 12 2 O r c h i d f l o w e r diagrams 2 0 3 F r o n t and s i d e - v i e w of H a b e n a r i a , l o n g i t u d i n a l s e c t i o n o f column i n H a b e n a r i a s p e c i e s used 2 1 4 Development of a n t h e r and o v u l e i n a p e r e n n a t i n g bud, showing next y e a r ' s f l o w e r s p i k e *^ 5 M i c r o s p o r o g e n e s i s , the m e i o t i c d i v i s i o n s ^7 6 M i c r o s p o r o g e n e s i s c o n t i n u e d , ^° 7 M i c r o s p o r o g e n e s i s , p o l l e n tubes ^9 8 - M i c r o s p o r o g e n e s i s , c o n t i n u e d ^ 0 4 l 9 Megasporogenesis i n H. hyperborea  ho 1 0 Megasporogenesis i n H. s a c c a t a  11 Megasporogenesis i n _ H . d i l a t a t a ^ 12 Megagametogenesis 1 3 Megagametogenesis, breakdown i n the female gameto- ^ phyte 14 Megagametogenesis, mature embryo sa c s ^ 47 15 / F e r t i l i a a t i n n 48 16 F e r t i l i z a t i o n , c o n t i n u e d 4? 17 Embryology — • 5C 18 Embryology, c o n t i n u e d ^ 51 19 Embryology c o n t i n u e d ., 2 0 Mature embryos ^ 2 1 Polyembryony " 2 2 - 2 3 Chromosome counts 6 5 A C K N O W L E D G M E N T . I t i s w i t h g r a t i t u d e t h a t I w i s h to acknowledge the h e l p and a s s i s t a n c e Dr. Beamish has g i v e n me d u r i n g t h i s s t u d y and a t a l l times I have been a t the U n i v e r s i t y o f B r i t i s h Columbia. I a l s o want to thank my o t h e r t e a c h e r s , Dr. C o l e , Dr. Maze, Dr. Marchant, Dr. Person and Dr. S c h o f i e l d who have never f a i l e d t o h e l p when i t was needed. Above a l l I owe thanks to my husband who has s u p p o r t e d me i n my s t u d i e s , p r o v i d e d t r a n s p o r t a t i o n f o r a l l my c o l l e c t i n g t r i p s and has s u f f e r e d l o n g and p a t i e n t l y d u r i n g my y e a r s a t the u n i v e r s i t y . I N T R O D U C T I O N . H a b e n a r i a fcWilld. i s a v e r y l a r g e p o l y m o r p h i c genus c o m p r i s i n g a t l e a s t 500 s p e c i e s . A l t h o u g h m o s t l y t r o p i c a l w i t h 100 s p e c i e s i n I n d i a a l o n e (Hooker 189*0 , the genus has some members i n temperate r e -g i o n s o f Europe, A s i a and N o r t h A m e r i c a . J u s t a h a n d f u l o f s p e c i e s are pr e s e n t i n A l a s k a , G r e e n l a n d , I c e l a n d and the F a r o e s . I n N o r t h America t h e r e a r e 20 s p e c i e s and by f a r the l a r g e s t number o f them, and the s h o w i e s t , are found i n the E a s t . Western Canada p r o v i d e s a home f o r 11 s p e c i e s , among them H. d i l a t a t a ( P u r s h ) Hook.,H. hyperborea ( L . ) R. Br . and H. s a c c a t a Greene,which are the s u b j e c t s o f t h i s s t u d y . C o n s i d e r i n g the g r e a t v a r i a b i l i t y e n countered w i t h i n the genus, i t i s not s u r p r i s i n g t h a t complete agreement as to i t s taxonomic t r e a t m e n t has never been r e a c h e d . The name H a b e n a r i a goes back to the L a t i n word "habena", a s t r a p o r thong. I t was f i r s t a p p l i e d by L i n n a e u s (1759) t o a Jamaican p l a n t which he c a l l e d O r c h i s h a b e n a r i a . B e i n g aware o f an i n h e r e n t d i f f e r e n c e between the European genus O r c h i s and the Jamaican p l a n t ^ W i l l d e n o w (1805) c r e a t e d the new genus H a b e n a r i a W i l l d . and l o o s e l y c i r c u m s c r i b e d i t t o i n c l u d e a l l known h a b e n a r i a s . A few y e a r s l a t e r (l8l7) L.C. R i c h a r d c l e a r l y d i f f e r e n t i a t e d between the t r o p i c a l H a b e n a r i a w i t h l o n g s t i g m a t i c p r o j e c t i o n s and a n t h e r c a n a l s , and p l a n t s of more temperate r e g i o n s w i t h c u p - l i k e , concave s t i g m a s and no s t i g m a p h o r e s , by e s t a b l i s h i n g the genus P l a t a n t h e r a L.C. R i c h a r d f o r the l a t t e r . John L i n d l e y (1835) r e c o g n i z e d both new genera and s e v e r a l c l o s e l y r e l a t e d ones, f i t t i n g them a l l i n t o one t r i b e , Ophrydeae. Taxonomic c o n f u s i o n grew, as the number o f s p e c i e s i n c r e a s e d . I t was then t h a t George Bentham d e c i d e d t o r e - u n i t e the v a s t complex under Habenaria W i l l d . He o u t l i n e d h i s i d e a s at a meeting of the L i n n a e a n S o c i e t y ( l 8 8 l ) and e n l a r g e d on them i n h i s Genera P l a n -tarum which he, t o g e t h e r w i t h S i r J.D. Hooker, p u b l i s h e d i n 1 8 8 3 . He c r e a t e d a number of s e c t i o n s w i t h i n the conglomerate genus, the two l a r g e s t b e i n g P l a t a n t h e r a and H a b e n a r i a proper as s e c t i o n s 9 and 1 0 . American b o t a n i s t s f o l l o w e d Bentham's l e a d (Gray 183*+, Ames 1 9 0 8 and 1 9 1 0 , C o r r e l l 1 9 5 0 ) and our N o r t h American s p e c i e s are now a l l grouped under H a b e n a r i a W i l l d . Rydberg ( 1 9 0 1 ) , who r e v i s e d p a r t of the genus as r e p r e s e n t e d i n N o r t h America n o r t h o f M e x i c o , was much opposed t o the c o n s e r v a t i v e views of Bentham and a g a i n s p l i t the genus i n t o many s e g r e g a t e s , some of which may deserve o n l y v a r i e -t a l s t a t u s . On the o t h e r hand, K r a e n z l i n ( 1 8 9 1 - 9 3 ) , who p a i n s t a k i n g l y monographed the t r o p i c a l h a b e n a r i a s , g r a n t e d s p e c i e s s t a t u s o n l y to H. hyperborea, c o n s i d e r i n g a l l o t h e r s as mere v a r i e t i e s . Bentham's treatment of the genus i s now g e n e r a l l y a c c e p t e d i n N o r t h America (Ames 1 9 1 0 , 1 9 2 4 , C o r r e l l 1 9 5 0 , S z c z a w i n s k i 1 9 5 9 , H i t c h c o c k et a l 1 9 6 9 ) a l t h o u g h many European b o t a n i s t s adhere to the s e g r e g a t e genus P l a t a n t h e r a . I n s p i t e o f a l l taxonomic changes and upheavals the t h r e e s p e c i e s i n q u e s t i o n , H. d i l a t a t a , H. hyperborea and H. s a c c a t a have always been c l a s s i f i e d t o g e t h e r i n t r i b e Ophrydeae by the e a r l y b o t a n i s t s and r e c e n t l y under O r c h i d e a e , s u b t r i b e O r c h i d i n a e . A c c o r d i n g to Rydberg ( 1 9 0 1 ) , C o r r e l l ( 1 9 5 0 ) , S z c z a w i n s k i ( 1 9 5 9 ) and o t h e r s , these s p e c i e s are c l o s e l y r e l a t e d and form h y b r i d swarms under s u i t a b l e c o n d i t i o n s . On the o t h e r hand, l a r g e p o p u l a t i o n s seem to r e t a i n t h e i r i d e n t i t y year a f t e r y e a r , even when growing s i d e by s i d e . T h e r e f o r e i t seemed of i n t e r e s t to t e s t t h e i r i n t e r r e l a t i o n -s h i p s e x p e r i m e n t a l l y . These t e s t s e n t a i l e d s t u d y of morphology, of p o l l e n p r o d u c t i o n and of seed development of the 3 s p e c i e s as found i n n a t u r e , and comparison of the n a t u r a l p r o c e s s w i t h seed development a f t e r i n t e r s p e c i f i c p o l l i n a t i o n s , , L e a v i t ( 1 9 0 1 ) d i d the f i r s t e m b r y o l o g i c a l s t u d y on the genus, w o r k i n g w i t h H. t r i d e n t a t a , now known as H. c l a v e l l a t a ( Michx.) Spreng. and H. b l e p h a r i g l o t t i s ( W i l l d . ) Hook., both E a s t e r n s p e c i e s not known from B r i t i s h Columbia. In 1 9 0 9 Brown worked on H. o r b i c u l a t a ( P u r s h ) T o r r . and H. i n t e g r a ( N u t t . ) Spreng. The l a s t e m b r y o l o g i c a l work on the genus was done by Swamy ( 1 9 ^ 6 ) who i n v e s t i g a t e d n i n e s p e c i e s of I n d i a n h a b e n a r i a s , the most d e t a i l e d work bei n g done on H. p l a t y p h y l l a Spreng. and H. r a r i f l o r a A. R i c h . S i n c e then no e m b r y o l o g i c a l s t u d i e s have been performed and no w e s t e r n s p e c i e s have ever been c r o s s e d a r t i f i c i a l l y o r i n v e s t i g a t e d o t h e r than m o r p h o l o g i c a l l y . T h e r e f o r e t h e r e a r o s e an e x c e l l e n t o p p o r t u n i t y , not o n l y t o s t u d y the r e l a t i o n -s h i p s of t h r e e w e s t e r n s p e c i e s , but to add to the knowledge o f H a b e n a r i a embryology i n g e n e r a l . Modern c l a s s i f i c a t i o n of the s p e c i e s used i n t h i s s t u d y i s t o be found i n Table I . A p a r t i a l l i s t o f those synonyms which i n d i c a t e the c o n f u s e d r e l a t i o n s h i p s a m o n g the s p e c i e s i s i n c l u d e d . Three v a r i e t i e s of H. d i l a t a t a are p r e s e n t l y r e c o g n i z e d , d i f f e r e n t i a t e d by l e n g t h and shape of the s p u r . I have never encountered the extreme form of v a r . l e u c o s t a c h y s b e a r i n g a spur 2 cm l o n g , a l t h o u g h t h i s v a r i e t y i s sup-posed to be c o a s t a l and w e s t e r n ( C o r r e l l 1 9 5 0 ) . H. d i l a t a t a v a r . a l b i -f l o r a from the North and from the R o c k i e s , w i t h a s h o r t almost s a c c a t e spu: has not been found i n B r i t i s h C o l u m b i a . Most o f t e n I have e n c o u n t e r e d p l a n t s w i t h s p u r s 2-3 mm l o n g e r than the l i p , which f a l l s on the border l i n e between H. d i l a t a t a v a r . d i l a t a t a and v a r . l e u c o s t a c h y s . They a r e r e f e r r e d t o s i m p l y as H. d i l a t a t a i n the p r e s e n t s t u d y . As the range of v a r i a t i o n i s extreme i n the s p e c i e s , i t i s not always easy t o d i f f e r e n t i a t e between v a r i e t i e s which o f t e n approach each o t h e r . T a b l e I. C l a s s i f i c a t i o n of H. d i l a t a t a , H. hyperborea and H. s a c c a t a based on Ames ( 1 9 2 4 ) , D r e s s i e r and Dodson ( I 9 6 0 ) , H i t c h c o c k et a l ( 1 9 6 9 ) and S z c z a w i n s k i ( 1 9 5 9 ) . A p a r t i a l l i s t o f synonymy i n d i c a t e s the co n f u s e d r e l a t i o n s h i p o f the s p e c i e s i n q u e s t i o n . F a m i l y . C r c h i d a c e a e . -Subfamily: O r c h i d o i d e a e . T r i b e : C r c h i d e a g . S u b t r i b e : C r c h i d i n a e . Genus: " a b e n a r i a '••'il/'d. S p e c i e s : H. d i l a t a t a ( P u r s h ) "ook., F. hyperborea ( L . ) P. ~ r . , F. s a c c a t a Greene. 1. H. d i l a t a t a ( P u r s h ) Hook. lF24 v a r . d i l a t a t a . Synonyms: C r c h i s d i l a t a t a Pursh 1 8 1 4 , H. b o r e a l i s Cham. l S 2 8 , R. b o r e a l i s v a r . d i l a t a t a Cham. l 8 ? 6 , P l a t a n t h e r a d i l a t a t a l i n d l . ex B e d l f t 3 3 , E' ££££iiif l i n d l . 1835, P. hyperborea v a r . d i l a t a t a P chb.f. 1 0 5 1 , P. hyperborea v a r . c o n v - j l l a r i a e f o l i a Vrzl. l 8 9 9 , H. d i l a t a t i f o r m i s Pydb. l8Q7. L i m n o r c h i s d i l a t a t a Pydb. 1901, — ' d i l a t a t a v a r . l e u c o s t a c h y s ( l i n d l . ) .Ames 1910, £• l e u c o s t - chys l i n d l . 1 351 H • l e u c o s t a c h y s '" 7ats. i 8 6 0 , 2* hyperborea v a r . l e u c o s t a c h y s K r z l . 1899. £.* d i l a t a t a v a r . a l b i f l o r a (Cham.) C o r r e l l 1943, H. b o r e a l i s v a r . a l H f l o r a Cham. 1 8 2 8 . 2 - h y p e r b o r e a ( L . ) R.Er. l 8 l 3 Synonyms: O r c h i s hyperborea L. 1767, 0 . h u r o n e n s i s N u t t . l 8 l 8 , H* h u r o n e n s i s Spreng. 1 8 2 6 , H* b o r e a l i s v a r . v i r i d i f l o r a Cham. 1 8 2 8 , P l a t a n t h e r a hyperborea ( L . ) L i n d l . 1835, L i m n o r c h i s hyperborea °ydb. 1 9 0 0 . 3- H. s a c c a t a Greene 18^5. Synonyms: H. u r a c i l i s S. '-'ats. 1877, P l a t a n t h e r a s t r i c t a L i n d l . 1835, H. s t r i c t a Pydb. l8°-7, l i m n o r c h i s s t r i c t a Pydb. 19C0, M A T E R I A L S A N D M E T H O D S . P l a n t s o f a l l 3 s p e c i e s were c o l l e c t e d d u r i n g J u l y and August o f 1 9 7 1 i a l l c o l l e c t i o n s b e i n g made i n B r i t i s h Columbia ( T a b l e I I , F i g . 1 ) . The p l a n t s were brought to the U n i v e r s i t y o f B r i t i s h C olumbia, p o t t e d i n a s u i t a b l e s o i l mix and i n s t a l l e d i n a c o l d frame. As the p l a n t s a r r i v e d , t h e i r morphology was s t u d i e d . Anther sqashes were made, which showed o n l y p o l l e n m i t o s e s , as m e i o s i s was past i n these months. O v a r i e s were p r e p a r e d f o r s e r i a l microtome s e c t i o n s and d u r i n g the w i n t e r megasporogenesis, gametogenesis, f e r t i l i z a t i o n and development o f the embryo i n the 3 s p e c i e s was o b s e r v e d . Over the w i n t e r months the p o t t e d p l a n t s were p r o t e c t e d w i t h a l a y e r of peat moss i n a c o l d frame and i n the s p r i n g o f 1 9 7 2 almost a l l emerged unharmed. M i c r o s p o r o g e n e s i s and development o f the p o l l e n g r a i n c o u l d then be f o l l o w e d and by mid-May the p l a n t s were ready f o r e m a s c u l a t i o n and p o l l i n a t i o n . The work had to be i n t e r u p t e d a t t h i s s t a g e , but was taken up a g a i n i n November. A l l p l a n t s seemed to have f l o w e r e d w e l l and s e t abundant seed. The p l a n t s were prep a r e d f o r a second w i n t e r and i n l a t e March and e a r l y A p r i l 1 9 7 3 i t was found t h a t more than a dozen had a p p a r e n t -l y not s u r v i v e d . Almost a l l of the H. s a c c a t a p l a n t s d i d not emerge and were presumed dead. T h e r e f o r e r e p l a c e m e n t s had to be c o l l e c t e d as soon as they c o u l d be l o c a t e d i n May. A few new H. d i l a t a t a p l a n t s were a l s o added. The f o l l o w i n g approach t o the p r o j e c t was adopted. 1 . A l l t h r e e s p e c i e s were used as seed p a r e n t s and r e c i p r o c a l c r o s s e s were made. 2. I n t r a s p e c i f i c c r o s s e s were made. , A few p l a n t s o f each s p e c i e s were emasculated and covered w i t h .5 • envelopes, to t e s t the t e c h n i q u e used. 4. A few p l a n t s o f each s p e c i e s were emasculated and l e f t open, un-p r o t e c t e d , t o see i f p o l l i n a t o r s were p r e s e n t . 5. F i n a l l y some p l a n t s were l e f t untouched and u n c o v e r e d . D u r i n g A p r i l and May of 1973 chromosomes of the 3 s p e c i e s were counted i n both a n t h e r c e l l s and r o o t t i p s . G l a c i a l a c e t i c acid-95% a l c o h o l (1:3) was used as a f i x a t i v e '.^ nd the a n t h e r squashes s t a i n e d w i t h p r o p i o n i c c a rmine. The r o o t t i p s were p r e t r e a t e d w i t h 2-alpha-bromo naphthalene at 45 F f o r 4 hours or more to c o n t r a c t the chromosomes. Root t i p s were then f i x e d as u s u a l , h y d r o l y z e d i n IN HC1 f o r 10 minutes and s t a i n e d a c c o r d i n g to the F e u l g e n r e a c t i o n . On May 16 the f i r s t c r o s s was made and from then onward, almost d a i l y , p l a n t s were c r o s s e d as they matured. The procedure was to emas-c u l a t e about h a l f the f l o r e t s o f a s p i k e , remove the r e s t o f the s p i k e and t r a n s f e r a whole p o l l i n i u m o f the same or a n o t h e r s p e c i e s t o each c u p - l i k e s t i g m a . A f t e r p o l l i n a t i o n each s p i k e was c overed w i t h a paper e n v e l o p e . To be e f f e c t i v e the p o l l i n i u m had t o be a t the r i g h t s t a g e of m a t u r i t y and t u r n i n g y e l l o w . I t s t i l l had to be unbroken i n t o "massulae" or p o l l e n p a c k e t s . I f massulae had become detached from the p o l l i n i u m when a bud was f o r c e d open, the f l o r e t had t o be d i s c a r d e d as s e l f -p o l l i n a t i o n might have ta k e n p l a c e , s t a r t i n g 3-4 days a f t e r p o l l i n a t i o n an ovary was removed from each h a n d - p o l l i n a t e d s p i k e at i n t e r v a l s f o r about 3-4 weeks and f i x e d i n FAA ( f o r m a l i n - a c e t i c a c i d - a l c o h o l , i n a r a t i o o f 6:4:90 of 50% a l c o h o l ) . S e r i a l microtome s e c t i o n s were p r e p a r e d i n the u s u a l manner. The s t a i n used was at f i r s t s a f f r a n i n - i r o n alum w i t h t a n n i n - o r a n g e G. L a t e r s a f f r a n i n - f a s t green was used e x c l u s i v e l y as i t gave b e t t e r c o n t r a s t and i s c e r t a i n l y a b e t t e r chromosome s t a i n f o r t h i s m a t e r i a l . O v a r i e s not removed f o r e m b r y o l o g i c a l s t u d i e s were l e f t on the s p i k e t o r i p e n n a t u r a l l y and when the c a p s u l e s s t a r t e d to open the powdery seed was c o l l e c t e d i n s m a l l paper envelopes and l e f t t o d r y . To e s t a b l i s h an approximate v a l u e f o r f e r t i l i t y o f a c r o s s the f o l l o w i n g procedure was adopted. Four o v a r i e s were chosen a t random from a s p i k e and the seed removed. From each l o t o f seed k e q u a l p o r t i o n s ( a s m a l l probe f u l l ) were mounted on a microscope s l i d e and 9 microscope f i e l d s examined i n each p o r t i o n . Ovules w i t h l a r g e w e l l formed embryos were c o n s i d e r e d f e r t i l e , empty ones s t e r i l e . The v a l u e s can be o n l y approximate as t h e r e were s l i g h t d i f f e r e n c e s i n embryo s i z e and amount of seed p i c k e d up on a probe. Some o v a r i e s con-t a i n e d l a r g e amounts of seed, whereas a few were almost empty. P e r c e n -tage o f v i a b l e seed t o the t o t a l number of seeds counted gave the mea-sure o f f e r t i l i t y . Voucher specimens o f the p l a n t s used i n t h i s s t u d y have been d e p o s i t e d i n the Herbarium o f 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 . T a b l e I I . C o l l e c t i o n l o c a t i o n s o f H. d i l a t a t a , H. h yperborea and H. s a c c a t a . A l l c o l l e c t i o n s i t e s were i n B r i t i s h C o l u m b i a . H. d i l a t a t a ( P u r s h ) Hook. Vancouver a r e a Ladner s a l t marsh No. 130 Manning P a r k Sumallo Lodge 1 0 1 O r c h i d Meadow 1 0 2 a O r c h i d T r a i l 124 A l l i s o n Pass v i c i n i t y 1 2 9 Squamish a r e a Erandywine F a l l s v i c i n i t y 1 0 6 " " No. 5 r o a d 1 0 8 W h i s t l e r a r e a ( 1 9 7 1 ) 1 1 0 " " ( 1 9 7 3 ) 1 3 2 Pemberton Meadows 1 1 8 Pemberton-D'Arcy 1 0 9 Mt. C u r r i e a r e a ( 1 9 7 1 ) 1 1 1 ji ( 1 9 7 3 ) 1 3 1 G a r i b a l d i P a r k T a y l o r C a b i n T r a i l 114 B l a c k Tusk Meadows 1 2 3 Keremeos Mt. Apex 1 1 2 B r a e l o r n e M c G i l l i v r a y Pass 1 2 2 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * H. hyperborea ( L . ) R.Br. Enderby Mara Meadows 1 1 6 , 1 1 7 Hunter's Range 137 Mt. T a t l o w Mt. T a t l o w Slope 1 2 5 Manning P a r k Ea s t o f Blow Down a r e a 1 0 3 , 1 0 4 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * H. s a c c a t a Greene. Manning P a r k O r c h i d Meadow . 1 0 2 b A l l i s o n Pass v i c i n i t y 1 0 5 Squamish a r e a Brandywine F a l l s 1 0 7 G a r i b a l d i P a r k T a y l o r C a b i n T r a i l 115 B r a e l o r n e M c G i l l i v r a y Pass 1 2 1 HarriF.nn Hemlock V a l l e y 1 1 1 , 133? 1 3 ^ i 1 3 6 Liumchen Liumchen V a l l e y 1 3 5 , 1 3 9 V . I . M a l l a r d Lake 1 2 8 S l o c a n Lake No. 8 Road 1 2 0 Kootenays J e w e l Lake 1 1 9 F i g u r e 1. Map of t h a t s e c t i o n o f B r i t i s h Columbia i n which c o l l e c t i o n s were made. Ik O B S E R V A T I O N S A N D D I S C U S S I O N S . I . HABENARIA MORPHOLOGY. The genus H a b e n a r i a has a p p a r e n t l y f o l l o w e d the g e n e r a l l i n e s o f o r c h i d e v o l u t i o n , always geared to r e d u c t i o n f o r the sake o f eco-nomy and to accommodation o f i n s e c t p o l l i n a t o r s ( D r e s s i e r 1 9 6 l , B r i e g e r 1 9 7 0 ) . The r e s u l t i n g o r g a n i z a t i o n i s h i g h l y e p g c i a l i z e d , encompassing s t r u c t u r e s and terms not a p p l i e d to o t h e r seed p l a n t s ( F i g s . 2 , 3 , 4 ) . The morphology o f o r c h i d s has been s t u d i e d i n d e t a i l by e a r l y o r c h i d o l o g i s t s (R. Brown 1 8 1 3 , H o f m e i s t e r 1 8 4 9 , G u i g n a r d 1 8 8 6 , S t r a s s b u r g e r 1 9 0 0 and o t h e r s ) . More r e c e n t l y S c h l e c h t e r ( 1 9 2 6 ) , Swamy ( 1 9 ^ 9 ) and B r i e g e r ( 1 9 7 0 ) habe d e a l t w i t h the s u b j e c t . The f o l l o w i n g d i s c u s s i o n on H a b e n a r i a morphology s e r v e s t o i n t r o d u c e the s t r u c t u r e s and terms i n use and i s based on the f i n d i n g s o f these w o r k e r s , supplemented by my own o b s e r v a t i o n s . The s e p a l s . The p e t a l o i d s e p a l s form the outermost c o v e r i n g o f the f l o w e r and are the l e a s t modified,, The p e t a l s . Of the 3 p e t a l s the median one has undergone the g r e a t e s t changes. I t has become the l i p o r l a b e l l u m and h e l p s to a t t r a c t i n s e c t s and t o guide them to n e c t a r and p o l l e n . As i t has become h e a v i e r and l a r g e r i t has brought on zygomorphy and a l s o r e s u p i n a t i o n , which i s a t w i s t i n g of the ovary by 1 8 0 degrees to b r i n g the l i p to the p o s i t i o n o f a co n v e n i e n t l a n d i n g p l a t f o r m f o r i n s e c t s . In the p r i m o r d e a l o r c h i d s the median p e t a l was uppermost, as i t i s s t i l l i n the bud of modern o r c h i d s b e f o r e anthesi6 ( F i g . 2 - A ) . A f t e r a n t h e s i s the f l o w e r becomes r e s u p i n a t e d as d e s c r i b e d above ( F i g . 2 - B ) . The s t a m i n a l w h o r l s . Monandrous o r c h i d s , i n c l u d i n g H a b e n a r i a , have o n l y one f u n c t i o n a l stamen, the median one of the o u t e r w h o r l . The column and p o l l i n i u n u In H a b e n a r i a as i n o t h e r o r c h i d s the column i s a f u s i o n o f stamens and s t y l e , the one f u n c t i o n a l stamen s e c u r e l y adnate t o the top of t h i s s t r u c t u r e . The v e r y s h o r t f i l a m e n t forms the attachment's of the a n t h e r t o the column base and c a r r i e s the s t e r i l e c o n n e c t i v e . The c o n n e c t i v e , s u p p l i e d w i t h the v a s c u l a r s t r a n d o f the a n t h e r , s e p a r a t e s the 2 a n t h e r c e l l s . W i t h i n each a n t h e r c e l l i s a p o l l i n i u m c o n s i s t i n g o f 2 p o l l e n masses. Each p o l l i n i u m c o m p r i s e s thousands of p o l l e n g r a i n s u n i t e d i n t o s m a l l p a c k e t s or "massulae" and h e l d t o g e t h e r more or l e s s f i r m l y by v i s c i n t h r e a d s . Compound p o l l e n g r a i n s are c h a r a c t e r i s t i c o f the o r c h i d f a m i l y as a whole and are found as t e t r a d s , as massulae i n p o l l i n i a as i n H a b e n a r i a , or j u s t as p o l l i n i a . Each p o l l i n i u m ends i n a s h o r t , s t i c k y , e l a s t i c s t a l k c a l l e d a c a u d i c l e , whic c o n s i s t s of m o d i f i e d p o l l e n g r a i n s . The c a u d i c l e i s f i r m l y a t t a c h e d to the v i s c i d g l a n d o r v i s c i d i u m which i s naked i n H a b e n a r i a . The c a u d i c l e s r e p r e s e n t male t i s s u e , whereas v i s c i d i a o r i g i n a t e from female s t i g m a t i c p a r t s . P o l l i n i u m i , c a u d i c l e and v i s c i d i u m t o g e t h e r are c a l l e d the p o l l i n a r i u m . Here a g a i n the t r e n d to r e d u c t i o n and c e n t r a l i z a t i o n i s apparent and shows the h i g h degree o f a d a p t a t i o n to i n s e c t p o l l i n a t o r s . Only, one i n s e c t v i s i t o r i s n e c e s s a r y t o c a r r y a whole p o l l i n a r i u m t o the next s t i g m a and to f e r t i l i z e a l l the o v u l e s o f i t s a t t a c h e d o v a r y . Because the p o l l e n g r a i n s have become u n i t e d , not a s i n g l e g r a i n i s wasted i f the p o l l i n a r i u m a r r i v e s s a f e l y on the next s t i g m a . T h i s ; " a l l or n o t h i n g " arrangement, p e r f e c t e d over l o n g p e r i o d s of t i m e , seems to have worked out w e l l f o r the O r c h i d a c e a e . The s t i g m a . -*-n H a b e n a r i a the s t i g m a i s a s h a l l o w c u p - l i k e d e p r e s s i o n con-s i s t i n g o f 3 s t i g m a t i c l o b e s , t h e i r l ower p a r t s f i r m l y grown t o g e t h e r and s i t u a t e d on the i n n e r s i d e of the column. I n more p r i m i t i v e o r c h i d s a l l 3 l o b e s are f e r t i l e , i n H a b e n a r i a p n l y the 2 l a t e r a l ones are f u n c t i o n a l . The median l o b e has become m o d i f i e d t o form the r o s t e l l u m , a g a i n a s t r u c t u r e found o n l y i n the O r c h i d a c e a e . I t i s a t i s s u e f l a p i n s e r t e d between the a n t h e r and the s t i g m a which h e l p s to p r e v e n t s e l f p o l l i n a t i o n . The v i s c i d i a , developed on the s t r a p - s h a p e d r o s t e l l u m , h e l p to b r i n g about c r o s s p o l l i n a t i o n by a t t a c h i n g themselves by means of a f a s t s e t t i n g cement t o a v i s i t i n g i n s e c t . They are a t t a c h e d to the r o s t e l l u m almost v e r t i c a l l y and l e a v e o n l y a f a i n t i m p r e s s i o n when removed. As the r o s t e l l u m i n the 3 s p e c i e s s t u d i e d has o n l y a s l i g h t m iddle f o l d , the v i s c i d i a are not f a r removed from each o t h e r . A l a r g e i n s e c t , f i t t i n g i n t o the s t i g m a t i c cup, can remove both.Ilnsecifes e n t e r i n g a f l o w e r and t o u c h i n g a v i s c i d i u m , c a r r y o f f one or both p o l l i n i a t o d e p o s i t them on the s t i g m a v i s i t e d n e x t . The p o l l e n germinates w i t h i n 24 hours i n the sugary s o l u t i o n produced by the s t i g m a . The v i s i b l e p a r t s of the s t i g m a t i c l o b e s seem to s w e l l and c l o s e a f t e r p o l l i n a t i o n . The p e r i a n t h d r i e s up and remains a t t a c h e d to the o v a r y . The o v a r y . The ovary i s i n f e r i o r , u n i l o c u l a r and composed o f 3 f u s e d c a r p e l s . The p l a c e n t a e are p a r i e t a l . The o v u l e s s i t u a t e d on the p l a c e n t a l r i d g e s a r e v e r y numerous and t i n y . B e i n g t e r r e s t r i a l , H a b e n a r i a e s t a r t s the development ojf the c u r r e n t y e a r ' s o v u l e s the p r e v i o u s summer ( F i g . 4-2). The i n t e r -v a l between p o l l i n a t i o n - f e r t i l i z a t i o n and the m a t u r a t i o n o f the embryo i s fyuch s h o r t e r than i n e p i p h y t i c o r c h i d s , where the o v u l e s are not i n i t i a t e d u n t i l p o l l e n i s p l a c e d on the s t i g m a (Swamy 194-9, B r i e g e r 1970). The stem. The stems o f h a b e n a r i a s are h o l l o w and each stem bears 6-8 s h e a t h i n g l e a v e s which become b r a c t s toward the apex. Each f l o r e t i s subtended by such a b r a c t , which has c o n s p i c u o u s s e r r u l a t e edges. B e i n g h o l l o w , t h e stem breaks e a s i l y i n the wind when not s u p p o r t e d by o t h e r v e g e t a t i o n . E s p e c i a l l y H. d i l a t a t a p l a n t s tend t o become t a l l and s p i n d l y . H e i g h t of the stem i s 20-120 cm or more. A f t e r m a t u r a t i o n o f the f l o w e r s p i k e , the stem d i e s down and o n l y the r o o t system o v e r w i n t e r s . The f l o w e r s p i k e . The s p i k e s are 8-40 cm i n l e n g t h and t h e r e i s g r e a t v a r i a t i o n i n the number o f f l o r e t s on a s p i k e , even w i t h i n the same s p e c i e s . Some s p i k e s are l a x , o t h e r s more dense. There can be as many as 100 f l o r e t s on a s p i k e , but u s u a l l y t h e r e a r e 20-40. The f l o w e r s open i n s u c c e s s i o n , s t a r t i n g w i t h the l o w e s t ones. At any time o n l y 4-6 f l o w e r s are open on a s t a l k , which i s one way o f p r e v e n t i n g p o l l e n o f the same p l a n t f a l l i n g on a s t i g m a lower down. Yet t h e r e are always some f l o w e r s open and ready s h o u l d an i n s e c t a r r i v e . D i f f e r e n c e s i n the i n d i v i d u a l f l o r e t s o f the 3 s p e c i e s are summarized i n T a b l e I I I . Roots and p e r e n n a t i n g buds. A l l members of the group possess a very s h o r t rhizome and f u s i f o r m , f i n g e r - l i k e r o o t s . O v e r w i n t e r i n g t a k e s p l a c e by means o f a y e a r l y d e v e l o p i n g a d v e n t i t i o u s underground bud ( F i g . 4 - 1 ) , which c o n t a i n s a l l o f next y e a r ' s i n f l o r e s c e n c e and the f o l i a g e l e a v e s . By the time the f l o w e r s p i k e i s i n f u l l bloom, a new bud i s formed near the o l d s t a l k . In v i g o r o u s p l a n t s more than one bud may d e v e l o p . D u r i n g the w i n t e r months the f o l l o w i n g y e a r ' s f l o w e r i n g s t a l k d e v e l o p s s l o w l y underground and when the s p i k e emerges i n e a r l y s p r i n g , the spore mother c e l l s i n the a n t h e r s a r e almost f u l l y developed ( F i g . 4 - 3 , 4 ) , so t h a t the m e i o t i c d i v i s i o n s f o l l o w i n r a p i d o r d e r . The o v u l e s have a l s o been i n i t i a t e d on the p l a c e n t a l r i d g e s ( F i g . 4 - 2 ) and when the p o l l e n i s a p p l i e d i n s p r i n g , f e r t i l i z a t i o n and the f o r m a t i o n o f the embryo w i l l take p l a c e i n a r e l a t i v e l y s h o r t time,, The s e e d s . H a b e n a r i a s p e c i e s l i k e a l l o t h e r o r c h i d s d e v e l o p t i n y bu numerous seeds. When r i p e the seeds c o n t a i n m o r p h o l o g i c a l l y u n d i f f e r e n t i a t e d embryos and l a c k endosperm. A m y c o r r h i z a l fungus i s needed f o r the g e r m i n a t i o n and e a r l i e s t n u t r i t i o n o f the seeds. The fungus which o f t e n i s a member of the R h i g o c t o n i a group e n t e r s the embryo v i a the suspen-s o r and i n f e c t s a number o f c e l l s n e a r e s t the s u s p e n s o r . Sometimes the embryo i s d e s t r o y e d by the fungus, but i n most cases a c e r t a i n b a lance between fungus and embryo i s r e a c h e d . H a b e n a r i a seems to outgrow i t s dependence on the symbiont i n mature s t a g e s , as t r a n s p l a n t a t i o n i s u s u a l l y s u c c e s s f u l . Of the m i l l i o n s o f seed produced i n an ovary o n l y a few f i n d a s u i t a b l e s u b s t r a t e , h a b i t a t and the i n v a d i n g fungus to be a b l e to germinate and to grow i n t o s e l f - s u p p o r t i n g p l a n t s . I I . MORPHOLOGY OF H. DILATATA, H. HYPERBOREA AND H. SACCATA. The three species are m o r p h o l o g i c a l l y s i m i l a r i n fea t u r e s j u s t described, but d i f f e r i n the f o l l o w i n g ways (Table I I I ) . The f l o r e t s . The v a r i a b i l i t y w i t h i n the three species i s always so great that characters tend t o overlap. Fragrance i n p a r t i c u l a r i s pronounced i n some members of a species and missing i n others. The flowers d i f f e r only s l i g h t l y i n s i z e , more i n co l o u r , shape of the l i p , l ength and shape of the spur, width of the connective, attachement of the anther c e l l s to the connective and shape of the v i s c i d i u m . The shape of the stem leaves, e s p e c i a l l y the lower ones, d i f f e r s , some being more rounded, others more acute. H. saccata has more b r a c t - l i k e sheathing bases near the s o i l l i n e . The v i s c i d glands of H. d i l a t a t a are shaped l i k e a human f o o t , i n the two other species they are round. Asa Gray stated that he would have followed K r a e n z l i n i n r e c o g n i z i n g only H. hyperborea as a good spe-c i e s , were i t not among other things f o r the d i f f e r e n c e i n the shape of the glands i n the 2 species (Gibson 1 9 0 5 ) . Attachement of the anther c e l l s to the connective i s a l s o s p e c i f i c . In H. d i l a t a t a the anther c e l l s are p a r a l l e l t o each other and the connective between them i s narrow. In H. saccata they are a l s o p a r a l l e l , but the connective i s wide. In H. hyperborea the anther c e l l s are d i v e r -gent toward the base of the anther. Figure 2. Orchid Flower Diagrams. Figure A and B. T y p i c a l monandrous o r c h i d flowers w i t h d o r s a l -v e n t r a l symmetry and 5 whorls of parts. A Flower not resupinated, d o r s a l sepal toward a x i s of i n f l o r e s c e n c e before anthesis. B Resupinated flower a f t e r anthesis. S i S2 l a t e r a l sepals; d o r s a l sepal. P i P2 l a t e r a l p e t a l s ; labellum. A j median stamen of outer whorl, the only f u n c t i o n a l one present i n Habenaria. B2 l a t e r a l stamens of inner whorl, present only as v e s t i g i a l s t r u c t u r e s . Cj C2 f u n c t i o n a l stigmas; s t i g m a t i c lobe modified i n t o r o s t e l l u m . St. Stem adapted from Brieger (1970). F i g u r e J>. F r o n t view (A) and s i d e view (B) of H a b e n a r i a f l o w e r , a = a n t h e r sac b = b r a c t co = c o n n e c t i v e 1 = l i p o = o v a r y , r e s u p i n a t e d i n B p = l a t e r a l p e t a l s r = r o s t e l l u m s = l a t e r a l s e p a l ds = d o r s a l s e p a l s t = s t i g m a sp = spur L o n g i t u d i n a l s e c t i o n of column i n H a b e n a r i a ( C ) . ( adapted from b r i e g e r 1970). c = c a u d i c l e co = c o n n e c t i v e It- f i l a m e n t p = p o l l i n i u m r = r o s t e l l u m s t = s t i g m a v = v i s c i d i u m T a b l e I I I . D i f f e r e n c e s i n f l o r e t morphology and i n the b a s a l stem l e a v e s . Species H. d i l a t a t a Colour, size of f l o r e t s white, 8-15 mm H. hyperborea j yellowish-green, 3 3-10 mm H. hyperborea 4n Lip rhombic-lanceolate , 5—8 mm linear to tapering fleshy, 3-6 mm H. saccata deep green, upper variable approach-half of petals j ing dila t a t a jllp, white, 6-12 mm j A-8 mm S p u r var. d i l a t a t a same length as l i p , 5-8 mm f i l i f o r m , s l i g h t l y bent, 2/3 of l i p var. leucostahys 1-2 cm often twice as long as l i p var. a l b i f l o r a h a l f as long as l i p c y l i n d r i c , always shorter than l i p yellowish-green, 5-9 mm l i n e a r - e l l i p t i c , fleshy, A.5-7 mm saccate, often didymous, 2.5-4 mm F i g u r e 4 . Development of a n t h e r and o v u l e s from an a d v e n t i t i o u s underground bud, s e c t i o n e d i n l a t e August 1 9 7 3 , showing next y e a r ' s f l o w e r i n g s p i k e enveloped by stem l e a v e s . 1 L o n g i t u d i n a l s e c t i o n o f bud showing f l o w e r s p i k e and stem l e a v e s ( x 2 8 0 0 ) . 2 The immature a n t h e r showing sporogenous t i s s u e ( a r r o w ) b l o c k e d out i n massulae ( x 5 0 0 0 ) . 3 D e v e l o p i n g o v u l e s on p l a c e n t a l r i d g e s ( a r r o w ) , s m a l l spur (arrow) x 4 5 0 0 . 4 Massulae c o n t a i n i n g sporogenous t i s s u e ( x 5 0 0 0 ) . I I I . MICROSPOROGENESIS AND THE MALE GAMETOPHYTE. The onset o f the m e i o t i c d i v i s i o n s can be r e c o g n i z e d by the enlargement o f the spore mother c e l l n u c l e i ( F i g . 8 - 1 ) . M e i o s i s f o l l o w s the u s u a l p a t t e r n w i t h homologous chromosomes p a i r e d and showing c h i a s m a t a , the chromosomes a l i g n e d a t the equator i n meta-phase and p u l l i n g a p a r t i n anaphase and t e l o p h a s e ( F i g . 5 - 1 , 2 , 3 , 4 , 5 ) . The f i r s t m e i o t i c d i v i s i o n produces a dyad. No w a l l s are l a i d down d u r i n g t h i s d i v i s i o n . The second d i v i s i o n i s s i m u l t a n e o u s i n the n u c l e i of the dyad, r e s u l t i n g i n a t e t r a d o f m i c r o s p o r e s , s t i l l w i t h i n the w a l l s of the spore mother c e l l ( F i g . 8 - 2 ) . Only a f t e r the second d i v i s i o n , when the n u c l e i have gone i n t o a r e s t i n g s t a g e , are c e l l w a l l s l a i d down by. i n w a r d l y d i r e c t e d f u r r o w s ( F i g . 8 - 2 ) . The r e s u l t i n g h a p l o i d m i c r o s p o r e t e t r a d s are as a r u l e i s o b i l a t e r a l ( F i g . 5 - 6 ) . or t e t r a h e d r a l ( F i g . 8 - 3 ) , but on o c c a s i o n a l i n e a r row of s p o r e s i s formed ( F i g . 8 - 4 ) i n massulae l o c a t e d i n the n a r r o w i n g end o f the a n t h e r . A l l the c e l l s o f one massula d i v i d e s i m u l t a n e o u s l y and so remain s y n c h r o n i z e d i n development ( F i g . 8 - 1 , 2 , 3 , 4 ) . Other massulae a r e i n a p p r o x i m a t e l y the same s t a g e . As the w a l l s o f the sp o r e s w i t h i n a massula a r e t h i n , t h e r e can be i n t e r a c t i o n between spore n u c l e i , so t h a t even chr o m o s o m e - d e f i c i e n t ones can d i v i d e ( B a r b e r 1 9 4 2 ) . T h i s phenomenon e x p l a i n s why a p p a r e n t l y no dead p o l l e n g r a i n s have been found i n the Or c h i d a c e a e ( B a r b e r 1 9 4 2 ) . The w a l l around the p o l l e n p a c k e t s i s c u t i n i z e d and does not a l l o w i n t e r a c t i o n of n u c l e i o f d i f f e r e n t massulae. A f t e r a p e r i o d o f r e s t the f i r s t d i v i s i o n of the m i c r o s p o r e s ( F i g . 6 - 1 ) , aggregated i n t e t r a d s and massulae, r e s u l t s i n the 2 - n u c l e a t e p o l l e n g r a i n ( F i g . 6-2) or male gametophyte w i t h i n the a n t h e r . T h i s d i v i s i o n i s c h a r a c t e r i z e d by a p r o l o n g e d prophase, the chromosomes c o n t r a c t i n g s t r o n g l y and becoming v e r y round and w i d e s p r e a d w i t h i n the c e l l . T h i s i s a f a v o u r a b l e p e r i o d f o r chromo-some c o u n t s . D i v i s i o n s i n the t e t r a d s are s i m u l t a n e o u s and s y n c h r o n i z e d ( F i g . 5-6) and a l a r g e v e g e t a t i v e and s m a l l e r , but deeper s t a i n i n g g e n e r a t i v e n u c l e u s i s formed ( F i g . 6-2). A c l e a r a r e a i n the c y t o -plasm can be seen s e p a r a t i n g the 2 n u c l e i though a c e l l p l a t e i6 l a i d down a t the time of the l a s t d i v i s i o n . A t r a n s i t o r y w a l l i s formed between the 2 c e l l s a t t h e i r f o r m a t i o n w h i c h , however, soon d i s -appears (Swamy 194-9). The g e n e r a t i v e n u c l e u s i s a t f i r s t l e n t i l - s h a p e d and surrounded by v e r y l i t t l e c y t o p l a s m ( F i g . 6-2). E v e n t u a l l y i t i s e n g u l f e d by the much g r e a t e r amount of c y t o p l a s m o f the v e g e t a t i v e n u c l e u s and i s f o r c e d i n w a r d , away from the c e l l w a l l . T h i s s t a g e c o n s t i t u t e s the almost mature p o l l e n g r a i n as i t i s shed. When brought upon a r e c e p t i v e s t i g m a i t germinates w i t h i n 24 h o u r s . At the time of s h e d d i n g the p o l l e n g r a i n has 2 w a l l l a y e r s ( F i g . 6-3). When the g r a i n germinates the i n t i n e pushes through the e x i n e and becomes the p o l l e n tube ( F i g . 7-1). U s u a l l y the vegetative;In"u:o.leus^,;sifollowed by the g e n e r a t i v e n u c l e u s and the c y t o p l a s m o f the g r a i n , e n t e r s the l e n g t h e n i n g tube and both are c a r r i e d i n the tube t h r o u g h the v e r y s h o r t s t y l a r c a n a l i n t o the o v a r y . On the way the g e n e r a t i v e n u c l e u s d i v i d e s , p r o d u c i n g 2 gametes or sperm n u c l e i ( F i g . 7-2,3). They are o v a l i n shape and s t r e t c h e d , h a v i n g been f o r c e d through the narrow neck of the ovary ( F i g . 7-3). The 2 sperm n u c l e i seem to be surrounded by a f a i n t l a y e r o f c y t o p l a s m . The v e g e t a t i v e n u c l e u s g r a d u a l l y l o s e s i t a s t a i n a b i l i t y and becomes very hard to see. When the stream o f p o l l e n tubes a r r i v e s i n the o v a r y , i t d i v i d e s i n t o 3 p a r t s , each stream heading f o r one p l a c e n t a l r i d g e . Here the assembled tubes d i v i d e a g a i n , some f o l l o w i n g each s i d e of the r i d g e . Then one p o l l e n tube e n t e r s one o v u l e a l o n g the f u n i c u l u s and through the m i c r o p y l e , , r e l e a s i n g the sperm n u c l e i i n t o one s y n e r g i d , or more r a r e l y between the s y n e r g i d s . I have compared the p r o c e s s of m i c r o s p o r o g e n e s i s i n H. d i l a t a t a , H. h y perborea and H. s a c c a t a to t h a t i n o t h e r members of the f a m i l y , and f i n d t h a t the compound p o l l e n g r a i n i s common to a l l w i t h v e r y few e x c e p t i o n s . However, the degree o f adherence of the g r a i n s d i f f e r s i n the v a r i o u s t r i b e s . The t h r e e w e s t e r n h a b e n a r i a s conform very c l o s e l y t o o t h e r members of the t r i b e O r c h i d e a e (Ophrydeae), w i t h the t e t r a d s i n massulae or p o l l e n p a c k e t s , many massulae f o r m i n g one p o l l i n i u m (Heusser 1914, Swamy 1 9 4 6 ) . On the o t h e r hand Tuschnjakova's s t u d y ( 1 9 2 8 1 ) on L i s t e r a o v a t a r e v e a l s the adherence of the p o l l e n g r a i n s i n t e t r a d s o n l y . L i s t e r a , b e l o n g i n g t o t r i b e N e o t t i e a e i s b e l i e v e d to be l e s s h i g h l y r e v o l v e d and i s u s u a l l y c l a s s e d b e f o r e the O r c h i d e a e ( D r e s s i e r & Dodson i 9 6 0 ) . A t h i r d comparison was made w i t h E u l o p h i a e p i d e n d r a e a (Swamy 1 9 4 2 ) , a genus b e l o n g i n g t o the h i g h l y e v o l v e d t r i b e Vandeae. Here the p o l l e n g r a i n s adhere i n complete p o l l i n i a , which i s the h i g h e s t s t e p i n adherence known and i n d i c a t e s h i g h s p e c i a l i z a t i o n . F i g u r e 5» M i c r o s p o r o g e n e s i s (The m e i o t i c d i v i s i o n s ) . 1 H. s a c c a t a . E a r l y prophase ( l e p t o t e n e - z y g o t e n e ) i n spore mother c e l l ( x 4 - 5 0 0 ) . 2 H. s a c c a t a . Metaphase I ( x 4500). 3 H. s a c c a t a . Anaphase I (x 4000). 4 H. s a c c a t a . Telophase I ( x 4500). 5 H. h y p e r b o r e a . Metaphase I I ( x 4 5 0 0 ) . 6 H. h y p e r b o r e a . S y n c h r o n i z e d p o l l e n m i t o s i s , i s o b i l a t e r a l t e t r a d s ( x 3 0 0 0 ) . F i g u r e 6. M i c r o s p o r o g e n e s i s ( c o n t i n u e d ) . 1 H. d i l a t a t a . S y n c h r o n i z e d p o l l e n m i t o s i s i n t e t r a d s a f t e r the m e i o t i c d i v i s i o n s ( x kOOO). 2 H. h y p e r b o r e a . Two-nucleate p o l l e n g r a i n , gn = gene-r a t i v e n u c l e u s , v | = v e g e t a t i v e n u c l e u s , ( x 3000). 3 H. d i l a t a t a . Two-nucleate p o l l e n g r a i n as i t i s shed. Heavy o u t e r w a l l l a y e r n o t i c e a b l e ( x 5000). Figure 7. Microsporogenesis ( p o l l e n tubes from p a r a f f i n s e c t i o n s ) . 1 H. hyperborea. Five days a f t e r p o l l i n a t i o n , p o l l e n tube showing v e g e t a t i v e and generative nucleus having reached the ovary. Arrows i n d i c a t e the 2 n u c l e i , (x 4000). 2 H. d i l a t a t a . Five days a f t e r p o l l i n a t i o n , generative nucleus (arrow) d i v i d i n g i n the p o l l e n tube, having reached the ovary, (x 4000). 2 H. d i l a t a t a . Five days a f t e r p o l l i n a t i o n , 2 sperm n u c l e i (arrow) i n the p o l l e n tube (x 4 0 0 0 ) . F i g u r e 8 . M i c r o s p o r o g e n e s i s ( c o n t i n u e d ) . 1 H. h y p e r b o r e a . Prophase of m i c r o s p o r e mother c e l l s i n massulae ( x 2 0 0 0 ) . 2 H. d i l a t a t a . F i n i s h e d t e t r a d of m i c r o s p o r e s s t i l l w i t h i n w a l l of mother c e l l ( x 3 0 0 0 ) . 3" H. s a c c a t a . P o l l e n m i t o s i s i n t e t r a d s , n = 2 1 , (x 3 8 0 0 , ) . 4 . H. s a c c a t a . L i n e a r t e t r a d of k m i c r o s p o r e s i n p o l l e n m i t o s i s , n = 2 1 , ( x 3 8 0 O ) . IV. MEGASPOROGENESIS AND THE FEMALE GAMETOPHYTE. Megasporogenesis a f t e r s u c c e s s f u l c r o s s e s i s so much a l i k e i n a l l 3 s p e c i e s t h a t one g e n e r a l i z e d d i s c u s s i o n i s adequate. The o v u l e s a r e i n i t i a t e d as m i c r o s c o p i c d i c h o t o m o u s l y b r a n c h i n g p a p i l l a e d e v e l o p i n g from the p l a c e n t a l e p i d e r m i s . The young o v u l e s c o n s i s t o f a l i n e a r row o f 5-6 c e l l s s urrounded by an e p i d e r m i s ( F i g . 10-2). The uppermost hypodermal c e l l i n t h i s row i s the a r c h e s p o r i a l c e l l ( F i g . 10-1) which d i f f e r e n t i a t e s d i r e c t l y i n t o spore mother c e l l s ' ( F i g . 9-D. The i n n e r integument appears e a r l y ( F i g . 10-3) and has surrounded the embryo sac by the time the megaspores are formed, l e a v i n g o n l y a s m a l l opening a t the m i c r o p y l a r end. I t i s 2 - l a y e r e d , whereas the o u t e r integument which d e v e l o p s below the i n n e r one a l i t t l e l a t e r , c o n s i s t s o f o n l y one l a y e r o f c e l l s . The o u t e r integument has outgrown the i n n e r one by the time the embryo sac i s mature. The f u n i c u l u s i s the l o w e s t p a r t o f the o v u l e and c o n n e c t s i t to the p l a -c e n t a . The o v u l e s are a t f i r s t o r t h o t r o p o u s , but soon become a n a t r o -pous. At the onset of the r e d u c t i o n d i v i s i o n s the n u c l e u s o f the megasporocyte moves to the upper p a r t o f the c e l l ( F i g . 10-2), en-l a r g e s and, g o i n g through the u s u a l d i v i s i o n s t a g e s ( F i g . 9-2,3» F i g . 10-3, F i g . 11-1), produces a dyad o f c e l l s ( F i g . 10-4, 1 1 - 2 ) . W a l l s are l a i d down d u r i n g megasporogenesis c o n t r a r y to the s i t u a t i o n i n m i c r o s p o r o g e n e s i s and the c h a l a z a l c e l l i s f a v o u r e d as t o s i z e ( F i g . 1 0 - 4 ) . The second d i v i s i o n may take p l a c e o n l y i n the lower c e l l o f the dyad or i n b o t h . In the f i r s t case the r e s u l t i s a row of 3 c e l l s ( F i g . 9-4, 1 1 - 3 ) . I n the second case both c e l l s o f the dyad d i v i d e to ><produce a l i n e a r row o f 4 megaspores, or q u i t e f r e -q u e n t l y a T-shaped group of c e l l s ( F i g . 1 1 - 2 ) , depending on the d i r e c t i o n of the s p i n d l e s of the dyad. In both cases o n l y the c h a l a z a l megaspore s u r v i v e s and becomes the f u n c t i o n a l embryo sac mother c e l l . T h e r e f o r e the embryo sac i s a monosporic one. Mega-s p o r o g e n e s i s i s a r a p i d p r o c e s s t a k i n g from 3 - 6 days i n i n t r a s p e c i f i c c r o s s e s and o f t e n a l i t t l e l o n g e r i n i n t e r s p e c i f i c ones . Growth of the f u n c t i o n a l megaspore i s v e r y e v i d e n t a f t e r the r e d u c t i o n d i v i s i o n s . I n the p r o c e s s the 2 or 3 n o n - f u n c t i o n a l megaspores d i s i n t e g r a t e ( F i g . 1 2 - 1 ) and are pushed t o g e t h e r , a p p e a r i n g as d a r k ' - s t a i n i n g r e d caps on top o f the young embryo sac i n s t a i n e d s e c t i o n s . Three .•suc.cfessi-ve^nuelear d i v i s i o n s ( F i g . 1 2-1 , 2,4) produce the monosporic, 8 - n u c l e a t e Polygonum-type sac ( F i g . 1 4 - 1 , 2 ) . A f t e r the f i r s t d i v i s i o n the 2 n u c l e i are s e p a r a t e d by a l a r g e v a c u -o l e . The next d i v i s i o n s are s i m u l t a n e o u s , or n e a r l y s o , and no w a l l s are l a i d down d u r i n g the d i v i s i o n s . From the 4 - n u c l e a t e s t a g e to matu-r i t y the m i c r o p y l a r n u c l e i are always l a r g e r than the c h a l a z a l ones and o f t e n d i v i s i o n s of the l a t t e r seem to be d e l a y e d ( F i g . 12 - 3 ) or do not take p l a c e ( F i g . 1 3 - 2 , 4 \ so t h a t e i t h e r 6 - or 7 - n u c l e a t e s a c s a r i s e . At t h i s s t a g e I have i n f r e q u e n t l y noted a v e r y l a r g e s p i n d l e i n the c h a l a z a l end of the sac i n s t e a d of the u s u a l two, i n d i c a t i n g a f u s i o n of the s p i n d l e s , the r e s u l t i n g n u c l e i presumably b e i n g d i p l o i d i n s t e a d o f h a p l o i d and l e a d i n g t o a 6 - n u c l e a t e s a c . A p p a r e n t l y the advance of the p o l l e n tube down the s t y l e a f f e c t s the r a t e of embryo sac development, s i n c e the m a t u r a t i o n o f the sac i s q u i c k e r when the p o l l e n tube and s t y l e are of the same s p e c i e s . A f t e r i n t r a s p e c i f i c p o l l i n a t i o n s a mature embryo sac can be found from 6 - 7 days from the time p o l l e n r e a c h e s the s t i g m a . A f t e r i n t e r s p e c i f i c p o l l i n a t i o n s i t may take from 8-l4 days or more b e f o r e the sac i s ready f o r f e r t i l i z a t i o n . Speed of development a f t e r p o l l i n a t i o n depends a l s o on the f r e s h n e s s of the p o l l e n , h e a l t h and v i g o u r o f the seed p a r e n t and s t a g e of m a t u r i t y of the f l o r e t s . The mature megagametophyte ( F i g . 14-1 , 2 ) comprises the egg appa-r a t u s , the two p o l a r n u c l e i and the f o u r n u c l e i at the c h a l a z a l end of the s a c . The two s y n e r g i d s are s i s t e r c e l l s . The s i s t e r nu-c l e u s of the egg, the p o l a r n u c l e u s , moves toward the c e n t e r of the embryo sac and i s a p p a r e n t l y not surrounded by a c e l l w a l l . The c h a l a z a l group a r r a n g e s i t s e l f as the t h r e e a n t i p o d a l s and the c h a l a z a l p o l a r n u c l e u s , which moves up to the o t h e r p o l a r n u c l e u s and u s u a l l y f u s e s w i t h i t b e f o r e f e r t i l i z a t i o n t a k e s p l a c e . The embryo sac keeps on e n l a r g i n g , s t i l l topped by the d i s i n t e g r a t i n g s p o r e s . The n u c e l l u s and most o f the i n n e r integument have been r e s o r b e d e a r l i e r . The a n t i p o d a l s , or whatever remains o f them, o f t e n s t a i n d a r k l y and b e g i n an e a r l y d e g e n e r a t i o n . In some cases they are s t i l l v i s i b l e a f t e r f e r t i l i z a t i o n . The p o l l e n tube e n t e r s the o v u l e v i a the m i c r o p y l e and i n v a d e s one of the s y n e r g i d s ( F i g . 16-1) which i s d e s t r o y e d . The p o l l e n tube u s u a l l y empties i t s c y t o p l a s m and the 2 sperm n u c l e i i n t o the one s y n e r g i d and from t h e r e the sperm makes i t s way toward the egg and the o t h e r toward the p o l a r n u c l e i . At times the p o l l e n tube empties i t s c o n t e n t s d i r e c t l y i n t o the s a c , a d v a n c i n g between the s y n e r g i d s ( F i g . 1 5 - 2 ) , both o f which then remain unharmed. The r e -s u l t i s the same i n both c a s e s . One sperm always seems t o move t o -ward the egg c e l l , whereas the o t h e r i s c a r r i e d toward the f u s i o n S U A l e u s ( F i g . 15-2). The sperms are now round and smaller than the egg c e l l . The contents of the sac are of t e n hard to see as f a t t y , dark-s t a i n i n g substances are brought i n t o i t by the ruptured p o l l e n tube ( F i g . 16-1.). At the time of f e r t i l i z a t i o n s t a r c h g r a i n s are of t e n present i n the c e l l , i n the f u s i o n nucleus and i n the one remaining synergid. Often one sperm can be seen c l o s e l y eppressed t o the egg c e l l ( F i g . 1 5 - 1 , 2 , 3 , ) , or already w i t h i n i t ( F i g . 15-2, 16-1,2 , 3 , ) and I have observed f u s i o n between egg and sperm nucleus i n various stages of completion ( F i g . 16 - 1 , 3 ) . The polar n u c l e i are most o f t e n fused before f e r t i l i z a t i o n ( F i g . 15~2), at times only p a r t i a l l y so (F i g . 15~3J 16-2). They are o f t e n c l o s e l y accompanied by the sperm (F i g . 15~3)• I** t r i p l e f u s i o n has taken place one can o f t e n see 3 n u c l e o l i w i t h i n the primary endosperm nucleus ( F i g . 17-2, 18-2). I f the p o l a r n u c l e i fuse e a r l y the f u s i o n nucleus becomes very l a r g e , o f t e n l a r g e r than the egg c e l l / As a r u l e t r i p l e f u s i o n does take place i n the three species studied. However, the t r i p l o i d endosperm nucleus u s u a l l y begins t o show signs of d e t e r i o r a t i o n soon a f t e r f e r t i l i z a t i o n . I t becomes crescent-shaped ( F i g . 17 - 1,2 , 3 ) and s t a i n s i n .the same way as the d i s o r g a n i z i n g a n t i p o d a l s . On occasion the -endosperm nucleus, a n t i p o d a l s and one synergid are i n good c o n d i t i o n a f t e r the d i v i s i o n s i n the zygote have s t a r t e d . The f e r t i l i z e d egg, the zygote ( F i g . 17-1), grows c o n s i d e r a b l y a f t e r f e r t i l i z a t i o n and r e s t s a few days before onset of the next d i v i s i o n s . The f i r s t d i v i s i o n of the zygote i s always t r a n s v e r s e , producing a 2 - c e l l e d proembryo ( F i g . 17-2 , 3 ) , c o n s i s t i n g of the micropylar or b a s a l , and the c h a l a z a l or t e r m i n a l c e l l . The next d i v i s i o n i s i n the b a s a l c e l l , r e s u l t i n g i n a 3 - c e l l e d proembryo ( F i g . 18-1), adding a middle c e l l . Next the t e r m i n a l c e l l d i v i d e s by a v e r t i c a l w a l l ( F i g s . 17-4, 18-1, 19-1). The next d i v i s i o n can be e i t h e r i n the t e r m i n a l or b a s a l c e l l , i n i t i a t i n g i n the l a t t e r case the suspensor which develops i n a l l h a b e n a r i a s and w i l l be d i s c u s s e d l a t e r . I n the 4 - c e l l e d proembryo 4 d i v i s i o n s f o l l o w , 2 i n the t e r m i n a l c e l l s and 2 i n the middle c e l l , p r o d u c i n g 2 t i e r s of 4 c e l l s each ( F i g . 18 - 3 ) . The next d i v i s i o n s produce 8 c e l l s i n each of t h e s e t i e r s t o form the o c t a n t s t a g e . The t i e r s o r i g i n a t i n g from the middle c e l l d i v i d e a g a i n p r o d u c i n g a l l the c e l l s o f the m i c r o p y l a r end of the o v a r y . F i n a l l y , d i v i s i o n s i n the d i s t a l end r e s u l t i n groups of much s m a l l e r c e l l s by f o r m a t i o n o f v e r t i c a l , t r a n s v e r s e and o b l i q u e j w a l l s . The c e l l s o f the upper p a r t o f the em-bryo a r e always l a r g e r than the r e s t and can be r e c o g n i z e d as the descendants of the m i d d l e t i e r ( F i g . 2 0 - 2,4). From the 5 - c e l l e d s t a g e ( F i g . , 1 9 - 2 , 5,4) d i v i s i o n s are i r r e g u l a r and hard to f o l l o w , but u l t i m a t e l y the mature embryo ( F i g . 2 0 -1,2,4) c o n s i s t s o f 5 0 - 6 0 m o r p h o l o g i c a l l y u n d i f f e r e n t i a t e d c e l l s . The t e r m i n a l c e l l a t a l l times c o n t r i b u t e s most to the mature embryo. The middle c e l l p r o -duces the uppermost l a y e r s and the suspensor does not c o n t r i b u t e to the f o r m a t i o n of the embryo p r o p e r . The suspensor and i t s f u n c t i o n . The d i v i s i o n s of the suspensor may s t a r t when the t e r m i n a l c e l l i s s t i l l u n d i v i d e d ( F i g s . 17-3, 1 8 - 2 ) or l a t e r , but are u s u a l l y f i n i s h e d b e f o r e the embryo i s f u l l y d e v e l o p e d . A l l d i v i s i o n s are t r a n s v e r s e and a row of 6-7 c e l l s emerges, whic h , as the c e l l s l e n g t h e n , pushes out of the embryo sac ( F i g . 17-4, 2 0 - 1 ) , the l a s t c e l l implanting i t s e l f i n the placenta ( F i g . 2 0 - 2 ) and developing an h a u s t o r i a l f u n c t i o n . The other c e l l s presumably help i n the t r a n s -port of n u t r i e n t s to the embryo. O c c a s i o n a l l y a suspensor was seen which developed the usual number of c e l l s , but did not push out of the embryo sac ( F i g . 1 9 - 4 ) . In such a case the embryo proper d i d not seem t o develop f u l l y . When the embryo matures and s t a r c h and aleurone g r a i n s f i l l the c e l l s , the suspensor d r i e s up. Sometimes part of the suspensor can s t i l l be seen i n mature seeds. Polyembryony. Several times I have noticed two embryos i n a young embryo sac, one embryo always being smaller than the other ( F i g . 2 1 - 1 , 3 ) . However, i n mature seed w i t h w e l l developed embryos, at l e a s t a dozen seeds were found w i t h two embryos w i t h i n the seed coat, but both of the same s i z e . According t o Le a v i t ( 1 9 0 1 ) and Swamy ( 19^9) who found m u l t i p l e embryos i n other Habenaria species, these embryos were a r r i v e d at by cleavage of the f i r s t embryo.Only one embryo survived (Swamy 19^9) as no supernumerary embryos were found i n ma-ture seed. The m u l t i p l e embryos which were seen i n the present study ( F i g . 2 1 - 1 , 2 , 3 ) were apparently formed i n the same way and could be noti c e d very e a r l y i n the formation of the embryo ( F i g . 2 1 - 1 , 2 ) , but some of these reached maturity. In F i g . 2 1 - 2 the s p l i t t i n g o f f of the second embryo can be seen r i g h t below the suspensor i n i t i a l , but one embryo seems t o be d i s o r g a n i z i n g . Several times i n embryo sacs w i t h already w e l l advanced embryos a second p o l l e n tube was observed, c o n t a i n i n g 2 sperm n u c l e i ( F i g . 1 9 - 1 , 3 ) . Ovary w a l l and placenta. In the course of ovule development, p o l l i n a t i o n and f e r t i l i z a t i o n the placenta becomes hypertrophied and densely cytoplasmic. The sus-pensor becomes embedded i n t h i s r i c h s t o r a g e a r e a . When p o l l i n a t i o n has t a k e n p l a c e the ovary becomes a f r u i t , s w e l l s and l o s e s i t s green c o l o u r , the d r i e d up p e r i a a t h r e m a i n i n g a t t a c h e d . An u n p o l l i n a t e d ovary s t a y s green much l o n g e r , but does not mature and no s w e l l i n g can be seen. I n about 3-4 weeks a f t e r p o l l i n a t i o n the normal f r u i t i s mature and the seeds are ready t o be d i s p e r s e d . ( F i g . 2 0 - J ) . A c c o r d i n g to S e s h a g i r i a h ( 1 9 4 l ) the growth hormones or a u x i n s i n the p o l l e n tube and p o l l e n s t i m u l a t e the p l a s t i d s p r e s e n t i n the c e l l s o f the p l a c e n t a and ovary w a l l to s y n t h e s i z e c a r b o h y d r a t e s , which i n the absence o f endosperm, produce n u t r i e n t s n e c e s s a r y f o r o r d i n a r y growth and development o f the f r u i t and the seeds. The s e e d s . S i m i l a r to a l l o r c h i d seed, those produced by the t h r e e H a b e n a r i a s p e c i e s are very l i g h t , v e r y numerous and have m o r p h o l o g i c a l l y un-d i f f e r e n t i a t e d embryos. The o n l y food p r e s e n t i s packed i n the c e l l s of the o v o i d embryo ( F i g . 2 0 G n l y the outermost l a y e r of the o u t e r integument remains and becomes the t r a n s p a r e n t seed c o a t ; a l l : the o t h e r l a y e r s o f c e l l s are absorbed or d i s i n t e g r a t e . D u r i n g matu-r a t i o n the c e l l s o f the o u t e r integument l o s e t h e i r p r o t o p l a s t s and become t r a n s p a r e n t and r e t i c u l a t e . The embryo seems suspended i n the a i r - f i l l e d c a v i t y o f the s e e d - c o a t , and at tim e s a few c e l l s of the suspensor are s t i l l v i s i b l e . The u n u s u a l f e a t u r e s o f megasporogenesis and development o f the f e m g l a : gametophyte i n H a b e n a r i a s p e c i e s s t u d i e d are not w i t h o u t p r e c e d e n t . O r c h i d l i t e r a t u r e i n g e n e r a l ( R o l f e 1909, Poddubnaya-A r n o l d i I960, Swamy 1949) s t r e s s e s the f a c t t h a t the i n f l u e n c e o f the p o l l e n and p o l l e n tube i s n e c e s s a r y f o r the i n i t i a t i o n ecf the o v u l e s and t h a t the o v u l e s as a r u l e have not s t a r t e d t o d i f f e r e n t i a t e when the p o l l e n a r r i v e s on the s t i g m a . T h e r e f o r e t h e r e are l o n g d e l a y s between p o l l i n a t i o n and f e r t i l i z a t i o n and between f e r t i l i z a t i o n and the m a t u r a t i o n o f the embryo i n a l l e p i p h y t i c and t r o p i c a l o r c h i d s , the i n t e r v a l becoming l o n g e r w i t h p r o g r e s s i v e l y h i g h e r e v o l u t i o n o f the s p e c i e s i n q u e s t i o n . H a b e n a r i a s b e i n g t e r r e s t r i a l and not as hii g h l y e v o l v e d as the e p i p h y t e s , do not need the presence o f p o l l e n f o r the i n i t i a t i o n o f t h e i r o v u l e s . When i n v e s t i g a t i n g v e r y young buds w i t h the p o l l e n not y e t mature, i n w s i i a b ^ y I found o v u l e s i n the a r c h e s p o r ' i a l c e l l s t a g e . P o l l i n a t i o n had not y e t taken p l a c e , but o v u l e s had been i n i t i a t e d and a l i n e a r row of 5-6 c e l l s , t o p p e d by the a r c h e s p o r i a l c e l l c o u l d be seen i n s c r a p i n g s from a l i v i n g o v a r y . Even the s l i g h t bulge of the i n n e r integument was p r e s e n t . Development would p r o g r e s s and mature o v u l e s would r e s u l t w i t h o u t p o l l i n a t i o n , but no embryos would be p r e s e n t i f not f e r t i l i z e d a t a l a t e r s t a g e . A f t e r p o l l i n a t i o n development o f the embryo sac i s r a p i d , showing the spore mother c e l l s i n a l l s t a g e s o f the m e i o t i c d i v i s i o n s o n l y 2-3 days a f t e r p o l l e n has been a p p l i e d i n most o v a r i e s . R e d u c t i o n o f the female gametophyte i s a c h a r a c t e r i n h e r e n t i n the whole f a m i l y and h a b e n a r i a s are no e x c e p t i o n . T h i s has been commented on by the v a r i o u s w o r k e r s (W.H.Brown 1909, A f z e l i u s 1916, Swamy 19^ -9) who have d e a l t w i t h the O r c h i d a c e a e . I t i s u s u a l l y the group o f a n t i p o d a l s which i s re d u c e d . Another p e c u l i a r i t y o f the f a m i l y i s the s u p p r e s s i o n o f endo-sperm which i s the r u l e f o r most genera. T r i p l e f u s i o n does not take p l a c e i n a l l o r c h i d s and w i t h o u t i t the second sperm degenerates (Nawaschin 1 9 0 0 , S t r a s s b u r g e r 1 9 0 0 , Sw3my 1 9 4 9 ) . Only a few o r c h i d s , among them V a n i l l a p l a n i f o l i a (Swamy 1 9 4 9 ) and some C y p r i p e d i u m s p e c i e s (Pace 1 9 0 8 ) d e v e l o p some endosperm t i s s u e b e f o r e degenera-t i o n s e t s i n . The s i g n i f i c a n c e o f the l a c k of endosperm i s u n c e r t a i n and some b o t a n i s t s have assumed t h a t t h i s i s the rea s o n f o r the u n d i f f e r e n t i a t e d o r c h i d embryos (Coulter&. C h a m b e r l a i n 1 9 1 2 ) . O t h e r s b e l i e v e t h a t endosperm does not de v e l o p because the p r i m a r y endosperm n u c l e u s i s r e s o r b e d and used by the zygote i m m e d i a t e l y (Heusser 1 9 1 4 ) . But even o r c h i d s p e c i e s which d e v e l o p some endosperm have u n d i f f e -r e n t i a t e d embryos and the Podostomaceae have f u l l y d eveloped em-bryos and no endosperm development. As the suspensor has no p a r t i n the c o n s t r u c t i o n o f the embryo pr o p e r -in H a b e n a r i a , the embryos were c l a s s e d as b e l o n g i n g to the "Onagrad" type by Johansen ( 1 9 5 0 ) . The suspensor i t s e l f f a l l s i n t o type I I a c c o r d i n g to Swamy (19*4-9). I have compared the development o f t h r e e w e s t e r n h a b e n a r i a s to the I n d i a n s p e c i e s o f Swamy ( 1 9 4 6 ) and cannot f i n d any a p p r e c i a b l e d i f f e r e n c e i n microgametogenesis, megagametogenesis and embryology, except t h a t i n h i s s p e c i e s o n l y 2 days are n e c e s s a r y from p o l l i n a t i o n to f e r t i l i z a t i o n , whereas the Canadian p l a n t s take from 6 - 1 0 days. T h i s d i f f e r e n c e might be e x p l a i n e d by m i l d e r c l i m a t e and h i g h e r tempe-r a t u r e s . I t was observed i n the p r e v i o u s c e n t u r y ( G u i g n a r d 1 8 8 6 , H o f m e i s t e r 1 8 4 9 ) t h a t i n h i g h e r e v o l v e d o r c h i d s the i n t e r v a l between p o l l i n a t i o n and f e r t i l i z a t i o n and the m a t u r a t i o n o f the embryo i s a l o n g e r one than i n more p r i m i t i v e ugehe.ES. S c h n a r f ( 1 9 3 1 ) t a b u l a t e d AO the r e s u l t of h i s s t u d i e s i n ovule development and seed maturation i n the Orchidaceae. He gives a pe r i o d from 8 - l 4 days from p o l l i n a t i o n t o f e r t i l i z a t i o n f o r Habenaria species. These f i g u r e s correspond w i t h my r e s u l t s . A f t e r a l l crosses, i n t r a - or i n t e r s p e c i f i c , some ovules began development and f a i l e d . The f a i l u r e r a t e was much higher a f t e r i n t e r -than i n t r a s p e c i f i c p o l l i n a t i o n s as evidenced by seed data (Table V I ) . The study of megagametogenesis and embryology showed no c o n s i s t e n t p a t t e r n of degeneration, merely t h e t the process was slowed down a f t e r i n t e r -s p e c i f i c crosses, e s p e c i a l l y i n those where d i p l o i d s were crossed w i t h p o l y p l o i d s . However, there was some breakdown of c e l l s i n a l l crosses. Table V. Stages i n ovule development at given times from p o l l i n a t i o n t o m a t u r i t y a f t e r crosses were made. 1 day 2-6 days 6-14 days 10-21 days 21 days onward Germina- megaspore developing embryo sacs, mature sacs, f e r t i l i z a t i o n . zygotes, mature em-bryos, maturing seed, r i p e n i n g o v a r i e s , d i s p e r s i o n of seed. t i o n of p o l l e n mother c e l l s , dyads, t e t r a d s proembryos, embryos i n a l l stages up t o matu r i t y . g r a i n s i n massulae on the stigma. l i n e a r and T-shaped. Figure 9« Megasporogenesis i n H. hyperborea. 1 Megaspore mother c e l l (arrow) i n r e s t i n g stage, 2 days a f t e r p o l l i n a t i o n (x4000). 2 Megaspore mother c e l l (arrow) i n e a r l y prophase (leptotene-zygotene), 2 days a f t e r p o l l i n a t i o n (x 4000). 3 Megaspore mother c e l l (arrow) i n anaphase, 4 days a f t e r p o l l i n a t i o n (x 4000). 4 Three megaspores, the 2 upper ones degenerating, the c h a l a z a l one (arrow), f u n c t i o n a l , 4 days a f t e r p o l l i n a t i o n (x 4 0 0 0 ) . F i g u r e 10 Megasporogenesis i n H. s a c c a t a , 1 Two d e v e l o p i n g o v u l e s ( a r r o w s ) b e f o r e p o l l i n a t i o n a t a n t h e s i s , one c e l l i n d i v i s i o n ( x 4000). 2 Megaspore mother c e l l ( a r r o w ) b e f o r e p o l l i n a t i o n a t a n t h e s i s ( x kOOQ). 3 Megaspore mother c e l l ( a r r o w ) i n metaphase I , 6 days a f t e r p o l l i n a t i o n , i n n e r integume nt v i s i b l e ( x 2500). 4 Dyad a f t e r f i r s t m e i o t i c d i v i s i o n , 8 days a f t e r p o l l i n a t i o n ( x 3 0 0 0 ) . Figure 11. Megasporogenesis i n H. d i l a t a t a . 1 Megaspore mother c e l l (arrow) i n prophase, s i x days a f t e r p o l l i n a t i o n , inner integument c l e a r l y v i s i b l e , (x 5 0 0 0 ) . 2 T-shaped t e t r a d , the upper dyad c e l l has d i v i d e d by a v e r -t i c a l w a l l , s p i n d l e s t i l l v i s i b l e ; these 2 c e l l s seem to be degenerating. The 2 lower spores both i n t a c t , (x 2500). 3 Three megaspores, the two upper ones degenerating, c h a l a z a l one (arrow) f u n c t i o n a l , 6 days a f t e r p o l l i n a t i o n , (x 4000). F i g u r e 12. Megagametogenesis. H. hyperborea (4n) x H. d i l a t a t a . F i r s t d i v i s i o n o f embryo sac mother c e l l , 12 days a f t e r p o l l i n a t i o n , 2 d e g e n e r a t i n g s p o r e s t o p p i n g the sac (x 5000). H. s a c c a t a x H. hyperborea ( 4 n ) . Two-nucleate s a c , 2 d e g e n e r a t i n g s p o r e s on t o p , , n u c e l l u s s t i l l i n t a c t , i n n e r integument almost e n c l o s i n g embryo sac ( a r r o w ) , o u t e r i n t e g u -ment v i s i b l e ( o . i . ) , 6 days a f t e r p o l l i n a t i o n ( x 4000). H. hyperborea (4n) x H. d i l a t a t a . T h r e e - n u c l e a t e s a c , 7 days a f t e r p o l l i n a t i o n , the c h a l a z a l n u c l e u s has not d i v i d e d ( x 5000). H. d i l a t a t a x H. hyperborea ( 4 n ) . F o u r - n u c l e a t e s a c , the 2 m i c r o p y l a r n u c l e i i n the p r o c e s s of the next d i v i s i o n , one of the c h a l a z a l n u c l e i p a r t l y s e c t i o n e d o u t , these 2 c e l l s show no s i g n s of d i v i s i o n ( x 3000). F i g u r e 13. Megasporogenesis c o n t i n u e d , Breakdown i n the c e l l s o f the female gametophyte. 1 H. hyperborea (4n) x H. d i l a t a t a . Two-nucleate embryo sac showing e a r l y breakdown i n the c h a l a z a l c e l l ( a r r o w ) . One d e g e n e r a t i n g megaspore on top of s a c , 12 days a f t e r p o l l i n a t i o n ( x 4000). 2 H. hyperborea (4n) x H. d i l a t a t a . F o u r - n u c l e a t e s a c , 12 days a f t e r p o l l i n a t i o n , the l ower c e l l s i n b e g i n n i n g d i s o r g a n i z a t i o n . , (x 4000). 3 H. hyperborea (4n) x H. d i l a t a t a . F i v e - n u c l e a t e s a c . The 2 m i c r o p y l a r c e l l s have d i v i d e d , s p i n d l e s t i l l v i s i b l e , 2 c e l l s p a r t l y s e c t i o n e d o u t; i n the c h a l a z a l end o n l y 2 c e l l s v i s i b l e , o n l y one c e l l seems to have gone thEOUgh the second d i v i s i o n , 14 days a f t e r p o l l i n a t i o n (x 3500). F i g u r e 14. Megasporogenesis c o n t i n u e d . Almost mature s a c s . 1 H. hyperborea ( 4 n ) , i n t r a s p e c i f i c , 7 - n u c l e a t e embryo sac b e f o r e f e r t i l i z a t i o n , 1.6 days a f t e r p o l l i n a t i o n (x 5 0 0 0 ) . 2 H. hyperborea (4n) x H. d i l a t a t a . E i g h t - n u c l e a t e embryo sac b e f o r e f e r t i l i z a t i o n , 12 days a f t e r p o l l i n a t i o n . Four n u c l e i i n c h a l a -z a l end of s a c , p o l a r n u c l e i not y e t f u s e d ( x 5 0 0 0 ) . F i g u r e 1 5 - F e r t i l i z a t i o n . 1 H. h y p e r b o r e a , i n t r a s p e c i f i c . One w e l l p r e s e r v e d s y n e r g i d ( s ) , egg c e l l ( e ) w i t h sperm (a r r o w ) t o u c h i n g i t , p o l a r n u c l e i (p) not q u i t e f u s e d , second sperm (arrow) t o u c h i n g one p o l a r n u c l e u s ( x 3000). 2 H. hyperborea ( 4 n ) x H. d i l a t a t a . Two s y n e r g i d s ( s ) w i t h p o l l e n tube above l a r g e zygote ( z ) , egg and sperm n u c l e i t o u c h i n g (arrow) w i t h i n egg c e l l , 2 f u s e d p o l a r n u c l e i ( 2 a r r o w s ) , second sperm (a r r o w ) c l o s e t o egg c e l l , 16 days a f t e r p o l l i n a t i o n ( x 3 0 0 0 ) . 3 H. h y p e r b o r e a , i n t r a s p e c i f i c . L arge f e r t i l i z e d egg c e l l ( e ) , 3 f u s i n g n u c l e i below egg c e l l r e p r e s e n t i n g the p r i m a r y endosperm n u c l e u s ( a r r o w ) , a n t i p o d a l ( a ) , 9 days a f t e r p o l l i n a t i o n ( x 3000). 4 H. hyperborea, i n t r a s p e c i f i c . P o l l e n tube, one w e l l preserved synergid ( s ) , l a r g e zygote (z) w i t h s t a r c h g r a i n s end large nucleus; f u s i n g polars p a r t l y sectioned out (x 3 0 0 0 ) . F e r t i l i z a t i o n c o n t i n u e d . H. s a c c a t a x H. h y p erborea ( 4 n ) . P o l l e n tube ( p ) , sperm n u c l e u s f u s i n g w i t h egg n u c l e u s w i t h i n egg c e l l ( a r r o w ) , f u s i n g p o l a r n u c l e i w i t h second sperm a t t a c h e d ( a r r o w s ) , one a n t i p o d a l ( a ) , 18 days a f t e r p o l l i n a t i o n ( x 3000). H. h y p e r b o r e a A i n t r a s p e c i f i c . Egg and sperm f u s e d ( a r r o w ) , p o l a r n u c l e i (p) o n l y p a r t l y f u s e d , second sperm (a r r o w ) c l o s e to egg c e l l , t r i p l e f u s i o n has not y e t taken p l a c e , 9 days a f t e r p o l l i n a t i o n ( x 3000). H. d i l a t a t a , i n t r a s p e c i f i c . Egg n u c l e u s almost c o m p l e t e l y f u s e d w i t h sperm n u c l e u s ( a r r o w ) , 8 days a f t e r p o l l i n a t i o n (x 3000). 1 H. s a c c a t a , i n t r a s p e c i f i c . Large z y g o t e , endosperm n u c l e u s ( a r r o w ) c r e s c e n t -haped, b e g i n n i n g d e g e n e r a t i o n , 19 days a f t e r p o l l i n a t i o n ( x 3 5 0 0 ) . 2 H. hype r b o r e a , i n t r a s p e c i f i c . L arge t e r m i n a l c e l l ( a r r o w ) , b a s a l c e l l p a r t l y s e c t i o n e d out ( b ) . P r i m a r y endosperm n u c l e u s w i t h 3 n u c l e o l i ( a r r o w ) , 12 days a f t e r p o l l i n a t i o n ( x 3 5 0 0 ) . 3 H. hyperborea (2n) x H. s a c c a t a . T w o - c e l l e d proembryo, .degenerating p r i m a r y endo-sperm n u c l e u s ( a r r o w ) , 1 s y n e r g i d ( s ) and d i s o r g a n i -z i n g a n t i p o d a l s ( a ) , 19 days a f t e r p o l l i n a t i o n ( x 3 5 0 0 ) . 4 H. d i l a t a t a x H. s a c c a t a . F o u r - c e l l e d proembryo, t e r m i n a l c e l l has d i v i d e d by a v e r t i c a l w a l l ( arrow) and one of the c e l l s i n d i v i s i o n , 2n = 42, 22 days a f t e r p o l l i n a t i o n ( x 3 5 0 0 ) . 1 H. hyperborea ( 4 n ) x H. s a c c a t a . F o u r - c e l l e d proembryo, t e r m i n a l c e l l has d i v i d e d by a v e r t i c a l wall'( a r r o w ) , l a r g e f u s i o n n u c l e u s s t i l l i n g-ood shape ( a r r o w ) , 1 s y n e r g i d ( s ) , 2 J days a f t e r p o l l i n a t i o n . Slow d e v e l o p i n g h y b r i d embryo, ( x 3 0 0 0 ) . 2 H. d i l a t a t a i n t r a s p e c i f i c . F i v e - c e l l e d proembryo, d e g e n e r a t i n g endosperm n u c l e u s w i t h 3 n u c l e o l i ( arrow) i n d i c a t i n g t r i p l e f u s i o n , Ik days a f t e r p o l l i n a t i o n ( x 3 0 0 0 ) , 3 H. hyperborea ( 2 n ) x H. d i l a t a t a . T r a n s v e r s e s e c t i o n o f an embryo, showing one t i e r of c e l l s i n quadrant s t a g e , the c e l l s i n d i v i -s i o n , 12 days a f t e r p o l l i n a t i o n ( x 5 0 0 0 ) . 1 H. saccata x H. d i l a t a t a . F o u r - c e l l e d proembryo t e r m i n a l c e l l i n d i v i s i o n (arrow), primary endosperm nucleus (e) degenera-t i n g , an e x t r a p o l l e n tube w i t h 2 sperms(( 2 arrows) v i s i b l e , 15 days a f t e r p o l l i n a t i o n (x 4000). H. hyperborea x H. saccata. M u l t i - c e l l e d embryo w i t h suspensor, primary endo-sperm nucleus (e) crescent-shaped and p a r t l y covering the embryo; one c e l l of embryo i n d i v i s i o n , one nucleus of endosperm seems t o be i n prophase ? (arrow), 22 days a f t e r p o l l i n a t i o n (x 4 0 0 0 ) . H. hyperborea, i n t r a s p e c i f i c . F i v e - c e l l e d embryo, suspensor i n i t i a l i n prophase (arrow), endosperm nucleus degenerated, second p o l l e n tube w i t h 2 sperm (arrow) v i s i b l e (x 4000). H. hyperborea (2n) x H. d i l a t a t a . Many-celled embryo, suspensor w i t h 3 c e l l s has not yet pushed out of the embryo sac, endosperm nucleus degenerated (arrow), 18 days a f t e r p o l l i n a t i o n (x 4 0 0 0 ) . F i g u r e 2 0 . Mature embryos. 1 H. s a c c a t a , i n t r a s p e c i f i c . Embryo w i t h l o n g s u s p e n s o r , 19 days a f t e r p o l l i n a t i o n , (x 4000). 2 H. hyperborea (4n).:ix H. s a c c a t a . Mature embryo, suspensor i m p l a n t e d on p l a c e n -t a . P l a c e n t a l t i s s u e d i s i n t e g r a t i n g , 32 days a f t e r p o l l i n a t i o n ( x 3 0 0 0 ) . 3 H. s a c c a t a . i n t r a s p e c i f i c . Seed ready to be d i s p e r s e d , 32 days a f t e r p o l l i -n a t i o n , seed c o a t L s u r r o u n d i n g the embryo,(x 3 0 0 0 ) . Figure 21. Polyembryony 1 H. saccata x H. d i l a t a t a . One l a r g e proembryo, a second, much sm a l l e r embryo has s p l i t o f f and i s i n d i v i s i o n ; endosperm nucleus and 3 antipodals d i s i n t e g r a t i n g , 10 days a f t e r p o l l i n a t i o n (x 3000). 2 H. d i l a t a t a x H. hyperborea (2n). Two embryos with common suspensor. The s p l i t t i n g o f f apparently occurred very e a r l y i n development, the embryo on the l e f t degenerating; endosperm d i s o r g a n i z e d , 21 days a f t e r p o l l i n a t i o n (x 3000). 3 H_. hyperborea (4n) x H. saccata. One almost mature embryo and a second much smaller one, c o n s i s t i n g of 5 c e l l s o n l y , 32 days a f t e r p o l l i n a t i o n (x 3000). V. CHROMOSOME COUNTS AND CROSSES. R e s u l t s . H. d i l a t a t a , H. h y p e r b o r e a and H. s a c c a t a have 2 1 p a i r s o f chromosomes i n a l l but one of the p o p u l a t i o n s checked i n the p r e s e n t s t u d y . The one e x c e p t i o n was a t a l l g r e e n - f l o w e r e d popu-l a t i o n 1 0 3 ( 2-6) i n Manning Park , where p o l y p l o i d s (n = 4-2) grew among d i p l o i d s (n = 2 1 ) . Counts ar e p r e s e n t e d i n Table V. Both chromosome numbers have been r e c o r d e d p r e v i o u s l y , d i p l o i d s f r e q u e n t l y , (Humphrey 1 9 5 4 , T a y l o r and M u l l i g a n 1 9 6 8 ) and p o l y p l o i d s from n o r t h e r n a r e a s (Harmsen 1 9 4 3 , L 6 v e 1 9 6 8 and R i t c h i e 1 9 5 6 ) . I n I 9 6 9 F.C. Bent determined the chromosome number of 12 H a b e n a r i a s p e c i e s from Nova S c o t i a and l i s t s H.. hyperborea v a r . h u r o n e n s i s as h a v i n g 42 p a i r s o f chromosomes, which s h o u l d i n d i c a t e t h a t not o n l y f a r n o r t h e r n p l a n t s are p o l y p l o i d s as has been suggested p r e v i o u s l y (Harmsen 1 9 4 3 ) . However, no o t h e r t e t r a p l o i d s have been r e c o r d e d from s o u t h e r n B r i t i s h Columbia. The c r o s s i n g program was c a r r i e d but as o u t l i n e d under " M a t e r i a l s and Methods". R e s u l t s are d i s c u s s e d i n the same o r d e r and are p r e s e n t e d i n summary i n T a b l e s V I , V I I , V I I I , . a n d IX. 1 . I n t r a s p e c i f i c c r o s s e s produced many seeds w i t h l a r g e w e l l formed embryos. Mean f e r t i l i t y was 6 0 - 9 0 % which would a l l o w f o r a tremendous number of p o s s i b l e g e r m i n a t i o n s ( T a b l e V I ) . 2 . Of the s p i k e s which were emasculated and p r o t e c t e d a l l . but one d r i e d up c o m p l e t e l y , i n d i c a t i n g t h a t the t e c h n i q u e was e f f e c t i v e . On o n l y one f l o w e r i n g s p i k e a few o v a r i e s d i d s w e l l s l i g h t l y , s u g g e s t i n g t h a t some p o l l e n must have reached the s t i g m a , p r o b a b l y d u r i n g removal of the p o l l i n i a . 5 . Each of the s p i k e s on which f l o r e t s were emasculated but l e f t u n p r o t e c t e d produced a few seed pods, i n d i c a t i n g t h a t p o l l i -n a t o r s must have been p r e s e n t , but not v e r y e f f e c t i v e . The o v a r i e s o f one such s p i k e produced o n l y 6 . 6 % of seed w i t h embryos. I have noted f l i e s , bees and a g r e a t number of s m a l l g r e e n i s h s p i d e r s s c u r r y i n g i n and out o f the f l o r e t s when d i s t u r b e d and a p h i d s t o -ward the end o f the summer, which p r o b a b l y were the prey the s p i d e r s were p e r s u i n g . As s p i d e r s are c a r n i v o r o u s , p o l l e n and n e c t a r c o u l d not have been a g r e a t a t t r a c t i o n , but i n t h e i r wanderings i n and out of the f l o w e r s , they might have been a b l e to t r a n s f e r a few l o o s e p o l l e n p a c k e t s from one f l o w e r to the o t h e r and b r i n g about f e r -t i l i z a t i o n . I have been unable to see n i g h t - f l y i n g moth which are supposed t o be the u s u a l p o l l i n a t o r s o f H a b e n a r i a ( van der P i j l & Dodson 1 9 6 6 ) . k. P l a n t s o f the t h r e e s p e c i e s l e f t w i t h n e i t h e r emascu-l a t i o n nor c o v e r d i d not a l l behave a l i k e . H. d i l a t a t a and H. h y p e r -borea d e v e l o p e d l a r g e numbers of c a p s u l e s c o n t a i n i n g many seeds o f which ?8 % had mature embryos i n H. d i l a t a t a and 60.k % i n H. hyper-borea . H. s a c c a t a produced o n l y empty seeds ( T a b l e V I ) . 5 . R e s u l t s o f i n t e r s p e c i f i c c r o s s e s were not e n t i r e l y c o n s i s t e n t , even when the same 2 s p e c i e s were i n v o l v e d . However, some f a c t s emerge. a) I n r e c i p r o c a l c r o s s e s i t d i d not seem t o m a t t e r i n which d i r e c t i o n the c r o s s was made. However, i t became apparent t h a t some p l a n t s w i t h i n a s p e c i e s showed h i g h e r f e r t i l i t y than o t h e r s . Pla#fc 1 0 3 - 6 , one of the t e t r a p l o i d s , was a f a i l u r e both as a seed and a p o l l e n p a r e n t , whereas a s i s t e r p l a n t , 1 0 3 - 4 gave good r e s u l t s i n both d i r e c t i o n s when c r o s s e d w i t h another s p e c i e s ( T a b l e s V I I and V I I I ) . b) Data o f c r o s s e s between H. d i l a t a t a and H. hyperborea are t a b u l a t e d i n Table V I I . I n 7 c r o s s e s w i t h H. d i l a t a t a as seed p a r e n t the mean f e r t i l i t y was 62 % and p r o b a b l y would have been h i g h e r i f seed o f a l l the c r o s s e s had been .".available f o r s a m p l i n g . Two p r o -m i s i n g s t a l k s were l o s t by breakage i n s p i t e of s t a k i n g . One c r o s s ( 1 0 1 x 117) d i d not produce s u f f i c i e n t seed to be counted, p r o b a b l y because the p o l l e n p a r e n t was not a v e r y v i g o r o u s p l a n t . I n one c r o s s w i t h v e r y lov; percentage o f a p p a r e n t l y good seed, mixed p o l l e n o f the t e t r a p l o i d p l a n t s was used which i n c l u d e d p o l l e n o f p l a n t 1 0 3 - 6 . As mentioned above, t h i s p l a n t i s a v e r y poor p a r e n t and the poor r e s u l t o f c r o s s 1 1 2 a x 1 0 3 ( T a b l e V I I ) may be the r e s u l t o f h a v i n g i n c l u d e d t h i s p o l l e n i n the m i x t u r e . c) Data o f c r o s s e s between H. d i l a t a t a and H. s a c c a t a are shown i n Table V I I . Of the 3 c r o s s e s made, seed i s a v a i l a b l e o n l y from c r o s s 1 1 0 x 1 3 3 w i t h the low r e t u r n o f 2 5 %. However, c r o s s 1 2 2 x 1 2 1 ( T a b l e V I I ) showed l a r g e w e l l formed embryos i n the p a r a f f i n s e c t i o n s , i n d i c a t i n g t h a t the 2 s p e c i e s w i l l c r o s s under f a v o u r a b l e c i r c u m s t a n c e s and w i t h w e l l e s t a b l i s h e d p l a n t m a t e r i a l . d ) H. hyperborea and H. d i l a t a t a , w i t h the former as seed p a r e n t , were c r o s s e d 6 t i m e s and produced a mean o f 5 3 . 4 % of good seed ( T a b l e V I I I ) . H. hyperborea c r o s s e d w i t h H. s a c c a t a gave c o n s i s t e n t l y h i g h e r r e s u l t s ( T a b l e V I I I ) . e) Crosses between H. saccata and the 2 other species w i t h the former as seed parent gave i n c o n c l u s i v e r e s u l t s mainly because so l i t t l e seed was a v a i l a b l e f o r sampling. The reason f o r t h i s w i l l be discussed s h o r t l y . However i n 3 crosses w i t h H. hyperborea 63 % of good seed was harvested, which i s f a r b e t t e r than the r e s u l t s f o r crosses w i t h H. d i l a t a t a . Problems encountered. Breakage of the flower s t a l k s , even a f t e r s t a k i n g , was a constant problem. The flower pots had to be l i f t e d i n and out of the co l d frame and c a r r i e d back and f o r t h to my work area. The s t a l k s are t a l l , hollow and very f r a g i l e , e s p e c i a l l y those of H. d i l a t a t a . The 2 other s p e c i e s , which are not q u i t e so t a l l , d i d not break as f r e q u e n t l y . The l o s s of pl a n t s over w i n t e r posed a d d i t i o n a l d i f f i c u l t i e s . With the exception of a few specimens, H. saccata had to be replaced i n the s p r i n g of 1973. The new a r r i v a l s , having been r e c e n t l y d i s t u r b e d , were not as w e l l e s t a b l i s h e d and a c t i v e l y growing as the other p l a n t s . Furthermore, when they were ready f o r p o l l i n a t i o n , only stored p o l l e n was a v a i l a b l e except when t h e i r own p o l l e n was used f o r i n t r a s p e c i f i c crosses. P l a n t s p p o l l i n a t e d w i t h p o l l e n one month o l d or older were always slow i n development. Embryos took f u l l y 25-28 days to mature, but seemed of good s i z e . Here, too, breakage destroyed 3 s t a l k s so that the success of the crosses can only be judgedi.from the p a r a f f i n s e c t i o n s . Unfortunately a l l ovaries of the l a s t 3 crosses made with H. saccata as seed parent, were c o l l e c t e d f o r s e c t i o n i n g , l e a v i n g none f o r seed counts. P l a n t 1 2 1 , a H. s a c c a t a c o l l e c t e d i n 1 9 ? 1 , o f f e r s p r o o f t h a t t h i s s p e c i e s w i l l c r o s s as w e l l as any o t h e r , a t l e a s t when a r t i -f i c i a l l y p o l l i n a t e d i f the p l a n t s are w e l l e s t a b l i s h e d and a c t i v e -l y g r o w i n g . T h i s p l a n t was found entwined w i t h H. d i l a t a t a ( 1 2 2 ) and as the p l a n t s c o u l d not be s e p a r a t e d w i t h o u t damage, they were p l a n t e d t o g e t h e r and bloomed w e l l f o r the l a s t 2 summers. When r e c i -p r o c a l l y crossed' the seeds developed w e l l . Autogamy v e r s u s o u t c r o s s i n g . As d i s c u s s e d i n " M a t e r i a l s and Methods" ( c o n t r o l 5 , pg» 8", p l a n t s n e i t h e r emasculated nor p o r t e c t e d ) , H. d i l a t a t a and H. hype r -borea p l a n t s were v e r y p r o l i f i c i n seed s e t . The r e g u l a r i t y and o r d e r l i n e s s of the seed pods on the s p i k e s makes one t h i n k of s e l f -p o l l i n a t i o n . I t seems t h a t even v e r y e f f i c i e n t bees would miss a f l o r e t o c c a s i o n a l l y . Gray ( 1 8 6 2 ) b e l i e v e d t h a t H. hyperborea c o u l d be e i t h e r autogamous or o u t c r o s s i n g and H. d i l a t a t a seems to f o l l o w the same mode o f p o l l i n a t i o n i f p o l l i n a t o r s do not a r r i v e i n time and the s p o l l e n i s ready to be shed. I t seems t h a t a t a c e r t a i n s t a g e o f m a t u r i t y the p o l l e n p a c k e t s b e g i n to break l o o s e more e a s i l y and a g e n t l e wind o r a s l i g h t touch can remove them. O f t e n , f o r c i n g . open a bud r e v e a l e d f r e e p o l l e n p a c k e t s i n the f l o w e r and i n the s t i g m a . These p o l l e n masses may have germinated on the s t i g m a and may have f e r t i l i z e d some o v u l e s of a f l o w e r , whereas the< r e s t - o f •'the p o l l i n i u m c o u l d have been c a r r i e d o f f by an i n s e c t and f e r t i l i z e d a n o t h e r f l o w e r . Another p o l l i n a t o r c o u l d have brought p o l l e n to the f i r s t f l o w e r , so t h a t both s y s t e m s can be at work a t the same t i m e . I n H. hyperborea p o l l i n a t i o n can take p l a c e i n the bud (Hagerup 1 9 5 2 b ) , as the a n t h e r thecae d e h i s c e b e f o r e a n t h e s i s . A c c o r d i n g to the same au t h o r ( 1 9 5 2 a ) , H a b e n a r i a s p e c i e s i n the f a r North( ( F a r o e s , Greenland) are almost always s e l f - p o l l i n a t e d , as few i n s e c t s are to be found i n these wet and c o l d c l i m a t e s . For t h i s r e a s o n p l a n t s w i t h genes f o r autogamy have a b e t t e r chance f o r s u r v i v a l . Autogamy, which i m p a r t s o n l y the g e n e t i c make-up of one p a r e n t , may have been o f s p e c i a l v a l u e t o the s p e c i e s a t c e r t a i n t i m e s and i n c e r t a i n a r e a s , as from a few windblown see^ds a new p o p u l a t i o n may have a r i s e n when f i n d i n g a s u i t a b l e h a b i t a t . T h i s c o u l d be one o f the many f a c t o r s which have a s s i s t e d the sp r e a d o f the s p e c i e s from the South, f o l l o w i n g the r e t r e a t i n g i c e masses, t o the A r c t i c C i r c l e , always f a v o u r i n g wet h a b i t a t s . As genes f o r o u t c r o s s i n g were r e t a i n e d , even an o c c a s i o n a l c r o s s p o l l i n a t i o n among s p e c i e s u s u a l l y d e p r i v e d o f p o l l i n a t o r s , may have h e l p e d to r e t a i n v i g o u r and g e n e t i c p l a s t i c i t y . Because o f p l a s t i c i t y and v a r i a b i l i t y w i t h i n the s p e c i e s they have been a b l e to f l o u r i s h i n many d i f f e r e n t h a b i -t a t s , r a n g i n g from n o r t h e r n bogs and f o r e s t s t o s o u t h e r n mountain meadows and c o a s t a l s a l t marshes.which a g a i n must have a f f e c t e d t h e i r 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 and growth h a b i t s . H. s a c c a t a appears t o be s e l f - i n c o m p a t i b l e , as a r t i f i c i a l l y p o l l i n a t e d p l a n t s respond w e l l t o p o l l e n o f o t h e r s p e c i e s , but p l a n t s l e f t u n p o l l i n a t e d and u n p r o t e c t e d i n the c o l d frame produced o n l y empty seed ( T a b l e I X ) . L i k e w i s e , a v e r y v i g o r o u s f l o w e r s t a l k , broken i n t r a n s p o r t and l e f t to r i p e n n a t u r a l l y w i t h o u t a c c e s s to p o l l i n a t o r s produced o n l y c h a f f , though i n t h i s case the f a i l u r e to produce seed may have been due. to l a c k o f n u t r i e n t s r a t h e r than t o absence o f p o l l i n a t o r s . A c c o r d i n g to van der F i j i and Dodson ( 1 9 6 6 ) most o u t c r o s s i n g o r c h i d s have e v o l v e d c o m p l i c a t e d systems, both m o r p h o l o g i c a l and g e n e t i c , t q p r e v e n t s e l f - p o l l i n a t i o n , s e l f - s t e r i -l i t y b e i n g one, and the o pening of o n l y a v e r y few f l o w e r s at one time a n o t h e r . Both systems are o p e r a t i n g i n a l l 3 s p e c i e s examined. D i p l o i d y v e r s u s p o l y p l o i d y . The most i n t e r e s t i n g p l a n t s were c e r t a i n l y the members of the H. h yperborea complex. They came from 5 d i f f e r e n t p o p u l a t i o n s s c a t t e r e d o v e r s o u t h e r n B r i t i s h Columbia ( T a b l e : i l ) and, w i t h the e x c e p t i o n o f one group a l r e a d y mentioned, were t y p i c a l H. hyperborea p l a n t s w i t h l a x racemes and y e l l o w i s h - g r e e n f l o w e r s and the chromosome count of 2 1 p a i r s e s t a b l i s h e d f o r the s p e c i e s . The e x c e p t i o n a l g r e e n -f l o w e r e d group was c o l l e c t e d i n Manning Park' i n a v e r y wet r o a d -s i d e d i t c h , i n t e r m i x e d w i t h o t h e r weedy v e g e t a t i o n . The p l a n t s were about 2 f t t a l l , w i t h s t o u t stems and many-flowered racemes of deep-green f l o r e t s a l l d e l i c i o u s l y f r a g r a n t . I n t e r s p e r s e d among the t a l l e r p l a n t s were more f r a g i l e green ones, t o be mentioned a g a i n l a t e r ^ and some o r d i n a r y H. d i l a t a t a p l a n t s . In g r o s s morphology the s m a l l green p l a n t s d i f f e r e d from the l a r g e r ones o n l y i n s i z e . I t was not c o n c l u s i v e l y apparent u n t i l s p r i n g of 1 9 7 3 t h a t the p o p u l a t i o n of s t o u t green p l a n t s was t e t r a p l o i d w i t h 42 p a i r s o f chromosomes i n the c e l l s . T h i s count had a l s o been d e t e r -mined by Harmsen ( 1 9 ^ 3 ) , by R i t c h i e ( 1 9 5 6 ) and by Bent ( 1 9 6 9 ) , b u t not p r e v i o u s l y f o r s o u t h e r n B r i t i s h Columbia. The p l a n t s were a t f i r s t thought t o be g r e e n - f l o w e r e d d i l a t a t a s , but f u r t h e r s t u d y seemed to p l a c e the p o p u l a t i o n c l o s e r t o H. hyperborea where i t has been i n c l u ^ ded i n t h i s paper. H y b r i d o r i g i n o f the p o l y p l o i d s suggested i t -s e l f , as the l i p of the f l o r e t s o f t e n approaches more c l o s e l y t h a t o f H. d i l a t a t a ( T a b l e I I I ) , b e i n g v a r i a b l e i n each p l a n t examined. A l s o the upper, d i s t a l h a l f o f the p e t a l i s w h i t e , the lower h a l f i s green, and the l i p i s deep-green. The l i p i s f l e s h y as i n t y p i c a l H. h y p e r b o r e a . In some f l o r e t s the v i s c i d g l a n d s a l s o resemble those of H. d i l a t a t a , which makes the h y b r i d o r i g i n even more p r o b a b l e . As m e i o s i s i s r e g u l a r ( F i g . 2 2-3, 2 3-1) and the p l a n t s , w i t h the e x c e p t i o n o f 1 0 3 - 6 , seemed to c r o s s w e l l ( T a b l e V I I ) , a l l o p o l y p l o i d o r i g i n s u g g e s t d i t s e l f . The v e r y d i s t u r b e d h a b i t a t would f a v o u r the e s t a b l i s h m e n t o f a h y b r i d p o p u l a t i o n and an i n c r e a s e i n chromo-some number o f t e n goes a l o n g w i t h h y b r i d i t y , e s p e c i a l l y i n p e r -e n n i a l s (van der P i j l and Dodson 1969). N e v e r t h e l e s s the o r i g i n o f the p o p u l a t i o n i s s t i l l a m y s t e r y , as no t y p i c a l H. hy p e r b o r e a p l a n t s were found i n the imme-d i a t e a r e a . H. s a c c a t a as a p o s s i b l e p a r e n t was e x c l u d e d from the s t a r t on m o r p h o l o g i c a l grounds. H. d i l a t a t a was p r e s e n t and c o u l d s u p p l y the genes o f the one p a r e n t . What r o l e d i d the more f r a g i l e green p l a n t s p l a y i n the p o p u l a t i o n ? In c o n t r a s t to the t a l l green p l a n t s they are d i p l o i d s . Could they r e p r e s e n t the p r i m a r y h y b r i d ? P l a n t 1 0 4 - 2 , one of the s m a l l green p l a n t s , was c r o s s e d w i t h H. d i l a - t a t a and, to judge from the amount of seed produced ( 6 3 %), t h i s c r o s s was s u c c e s s f u l . The r e c i p r o c a l c r o s s was even more s u c c e s s f u l (71.5 % ) • M e i o s i s i n 104 - 2 i s r e g u l a r , a f a c t which c a s t s doubt on i t b e i n g the p r i m a r y h y b r i d , as a f e r t i l e h y b r i d i s not l i k e l y to p r o -duce a f e r t i l e p o l y p l o i d . As a whole the t e t r a p l o i d s were s u c c e s s f u l both as seed and as p o l l e n parentr, a g a i n w i t h the e x c e p t i o n o f p l a n t 1 0 3 - 6 . Some p l a n t s w i t h i n a s p e c i e s seem c o m p a t i b l e when c r o s s e d , w h i l e o t h e r s r e p e l f o r e i g n p o l l e n by e f f e c t i v e i s o l a t i n g mechanisms. The f a c t t h a t the s p e c i e s have remained d i s t i n c t , even when growing s y m p a t r i c a l l y may be r e l a t e d to t h i s o b s e r v a t i o n . In any case n a t u r a l h y b r i d s are not p l e n t i f u l and except f o r i n t e r m e d i a t e s i n t h i s p o p u l a t i o n , none were found. The q u e s t i o n remains where t o p l a c e t h i s s m a l l h y b r i d p o p u l a t i o n . As an i n t e r e s t i n g s i d e l i g h t to h y b r i d i t y i n the h a b e n a r i a s we f i n d i n Gray's New Manual of Botany ( F e r n a l d 1 9 0 8 ) the f o l l o w i n g e x c e r p t s under H a b e n a r i a Willdo H. media (Rydb.) N i l e s : "Growing w i t h H. hyperborea v a r . h u r o n e n s i s and H. d i l a t a t a and e v i d e n t l y a h y b r i d of them". U n f o r t u n a t e l y the chromosome number o f H. media i s not known and we do not have H. hyperborea v a r . h u r o n e n s i s i n our a r e a . On the o t h e r hand Ames makes the f o l l o w i n g comment under "Notes on H a b e n a r i a " ( 1 9 0 8 ) ; H. d i l a t a t a v a r . media (Rydb.) n.comb. ( L i m n o r c h i s media Rydb.) " An e x a m i n a t i o n of l a r g e q u a n t i t i e s has c o n v i n c e d me, t h a t Dr. Rydberg's L i m n o r c h i s media i s s i m p l y a v a r i e t y o f H. d i l a t a t a , c h a r a c t e r i z e d by y e l l o w - g r e e n f l o w e r s " . The parentage o f the Manning p o l y p l o i d p o p u l a t i o n i s not known, but on the b a s i s of morphology h y b r i d o r i g i n seems more p r o b a b l e than the p o p u l a t i o n b e i n g j u s t a g r e e n - f l o w e r e d v a r i e t y o f H. d i l a t a t a . Only one o t h e r s p e c i e s i s found i n the immediate s u r r o u n d i n g s , but l o n g d i s t a n c e d i s p e r s a l by wind o f the v e r y l i g h t seed i s common i n the O r c h i d a c e a e and H. hyperborea has been found i n o t h e r l o -c a t i o n s i n the P a r k . A few chance seeds of t h i s s p e c i e s may have reacheai-'ithe a r e a and f i n d i n g a s u i t a b l e h a b i t a t and a c o m p a t i b l e s p e c i e s , may have produced some h y b r i d o f f s p r i n g . I f such an event d i d take p l a c e , the o r i g i n a l p l a n t s may not have been found or may have d i s a p p e a r e d . P o l y p l o i d y o f t e n f o l l o w s h y b r i d i z a t i o n and r e s u l t s i n a l l o p o l y p l o i d s which are f e r t i l e , as a r e the Manning p l a n t s . T h i s f e r t i l i t y may go o n l y as f a r as the p o l y p l o i d g e n e r a t i o n and i t may be t h a t the p r i m a r y h y b r i d s from the seeds which I have produced by a r t i f i c i a l p o l l i n a t i o n are p a r t l y or c o m p l e t e l y s t e r i l e . The p e r e n n i a l h a b i t o f H a b e n a r i a a l l o w s f o r v e g e t a t i v e r e p r o d u c t i o n , so t h a t a s m a l l p o p u l a t i o n of even a few h y b r i d s can soon a r i s e . As back c r o s s i n g and i n t r o g r e s s i o n may be i n v o l v e d , m o r p h o l o g i c a l and p r o b a b l y g e n e t i c v a r i a b i l i t y may r e s u l t , to produce d i f f i c u l t i e s and c o n f u s i o n f o r the t a x o n o m i s t . As the h y p erborea i n f l u e n c e i n t h i s s m a l l p o p u l a t i o n seems s t r o n g e r than the d i l a t a t a c h a r a c t e r i s t i c s we may be j u s t i f i e d i n i n c l u d i n g t h i s group i n a H. hyperborea complex. I t would take a n o t h e r summer's work d e a l i n g w i t h j u s t these p l a n t s to t r y to s o l v e the problem. U n f o r t u n a t e l y , when the a r e a was v i s i t e d i n May of l a s t y e a r , a b u l l d o z e r had been through the d i t c h , w i p i n g out most of the v e g e t a t i o n , but h o p e f u l l y the p l a n t s w i l l grow a g a i n from t h e i r r o o t systems. Table V. Chromosome counts f o r H. d i l a t a t a , H, hyperborea and H. saccata, the species used i n t h i s study. 64a H. dilatata H. hyperborea H. hyperborea H. saccata n - 21 n - 21 n - 42 (4n) n - 21 101 Sumallo Lodge 104 Blow Down, Menning Perk 103 Blow Down, Manning Park 111 Hemlock Valley 102 Orchid Meadow Menning Park 116 Mara Meadow Enderby 105 A l l i s o n Pass Manning Park 112 Apex Kt.. Keremeos 117 Mara Meadows Enderby 119 Jewel Lake Kootenays j 118 Pemberton Meadows 120 Slocan Lake I Kootenays 1 124 Orchid T r a i l Manning Park 121 McGillivray Pass 125 Mt. Baker 1J0 Ladner s a l t -marsh 2n - 42 2n - 42 2n - 84 2n - 42 101 Sumallo Lodge 104 Blow Down Manning Park 1 0 J - 6 Blow Down Manning Park 105 A l l i s o n Pass Menning Park 112 Apex Mt. ' Keremeos 104-2 Blow Down Menning Park 135 Hemlock Valley Liumchen Val-ley • — 1 Counts l i s t e d as "n" were determined from pollen mother c e l l s in meiosis, those l i s t e d as "2n" from root t i p s . Figure 22. Chromosome counts made of. p l c n t s of H. d i l a t a t a , H. hyperborea and H. saccata . 1 H. d i l a t a t a , Metaphase I . / n = 21 ' (x 3CC0). . 2 H. saccata D i a k i n e s i s , n ^ 21 (>'- ACCO). 2 H, hyperborea (An) n c A2 (x 40C0)„ . Figure 2^. Chromosome counts continued, 1 H. hyperborea ( A n ) . Camera l u c i d a drawing from a n i r r e g u l a r cnther w i t h A p o l l e n sacs, a l l A c o n t a i n i n g c e l l s w i t h A2 p a i r s of chromosomes. 2 H. hyperborea ( d i p l o i d ) Root t i p count 2n - A2, ( s k e t c h ) , «\ myf *S * 1 / • • ® J* j 1 Table VI. Mean values of f e r t i l i t y i n crosses made. "Good" seed - seed w i t h large w e l l formed embryos. % of good seed - (supposed) f e r t i l i t y . Good seed x 100 •= % of f e r t i l i t y t o t a l seed counted "Good cross" - with many w e l l formed embryos i n the p a r a f f i n s e c t i o n s . CO H. d i l a t a t a " cr* ~ H. hyperborea H. saccata v - r 7 1 emasculated with cover emasculated open open p o l l i n a t e d I Notes K. d i l a t a t a o f intraspe-cif i c 66 % mean % of "pood" seed 62 % 50 % ? 1 good cross, no s~eed d r i e d up very few capsules 78 % "good" seed = seed with l a r g e w e l l forced erabrvos 2 crosses 4 crosses 2 crosses poor % of good eeed = f e r t i l i l (supposed' i n t r a s p e c i f i f c E. hycerborea O •+ 53.4 % 9? % 7 .^1 % d r i e d up very few capsules 60.4 % good seed x 100 = % t o t a l seed counted 6 crosses 2 crosses 2 crosses 6.6 % i n t r a s p e c i f i c H. saccata o t 31.1 % value f o r 1 o: 5 crosses onl; no other seed 63 % 4 crosses 2 "good" crosses, no seed d r i e d up extremely few pods 27 % "good" c r o s s = with numerous, w e l l formed embryos i n p a r a f f i n s e c t i o n s . Table V I I . C r o s s e s w i t h H. d i l a t a t a as seed p a r e n t . A l l p l a n t s d i p l o i d except 103 ( 4 n ) . Seed parent H. d i l a - t a t a . 102a l C 2 a - l 132-1 110-1 130-3 110-4 1 0 6 - 2 1 0 1 - 3 1 1 2 a - 3 112 a - 1 112b 1 3 0 - 3 1 0 9 - 2 110-3 132-2 122 P o l l e n parent open p o l l i -nations II* d i l a t a t a 110-4 1 3 0 - 3 H. hyperbo-rea 117 117 104-2 103 mixed pollen 1 0 3 - 6 103-4 103-4 1 saccata 133-3 133 121 No. of peeds counted with fmbryos with-out t o t a l 611 306 917 618 38 650 289 130 419 678 52 728 471 422 288 239 759 661 473 181 stqred p o l l 469 I 188 101 506 s t a l k 173 very j exc e l l f 276 173 broken, r 509 oor deve nt devel 654 1, no see'd. 657 307 679 o seed 682 oprr.ent c pment, st; 6 6 . 6 94 69 83 Penarks open p o l l i n a t i o n s 78 % mean 62 69 7 2 . 3 7 1 . 5 intrasnee i f i c 66 i n t e r s p e c i f i c ovules smaller than u s u a l . l a r g e embryos, pood cross 2 7 . 5 ji very l i t t l e seed. s t a l k broken no seed 7 4 . 5 s ovules w e l l deve-I loped. 3 s e c t i o n s show very good development. 62 % mean 25 l k broker J i n t e r s p e c i f i c . only c h a f f new plant,nopeed w e l l e s t a b l i s h e d pi ants arprox. 5056 T a b l e V I I I . C r o s s e s w i t h H. hyperborea as seed p a r e n t . A l l p l a n t s d i p l o i d except 103 ( A n ) . Seed F o l l e n No. of seeds counted % Perrarks parent parent with embryos with-out t o t a l f e r t i l i -ty H. hyperbo-rea 117-1 117-2 1 0 3 - 3 1 0 3 - 1 open p o l l i -nations 451 2 9 8 776 518 356 235 300 383 807 533 1 . 0 7 6 901 5 6 . 9 56 72 5 7 . 5 Ct>en p o l l i n a t i o n s p r o l i f i c seed set 6 0 . 4 % mean H. hyperbo-rea i n t r a s p e c i f i c 1 0 3 - 3 1 0 3 - 103x 633 19 652 97 p r o l i f i c , w e l l de-veloped seed 9 7 % mean H. d i l a t a t a i n t e r s p e c i f i c 116 mixed p o l l e n 448 307 7 5 5 5 9 . 5 117 1 1 2 a - l 129 268 397 32 week seed parent 104-2 1 0 3 - 4 1 0 3 - 4 1 1 2 a - 3 mixed p o l l e n mixed p o l l e n 472 588 278 130 3d develo 750 718 ;ment, nc 63 82 seed good embryos,same % ase as i n r e c i -p r o c a l c r o s s , w e l l formed embry-os s t a l k broken I 0 3 - 6 . 112b 243 546 789 31 >nly c h a f f , same r e -s u l t as r e c i p r o c a l cross 53.4 % mean H. saccata i n t e r s p e c i f i c 1 0 3 - 3 x 1 0 3 - 4 121 113 543 725 314 1 3 0 857 855 6 3 . 5 84 . 7 w e l l developed seed p r o l i f i c seed set, lar g e embryos. 7^ % mean Table IX. C r o s s e s w i t h H. s a c c a t a as seed p a r e n t . A l l p l a n t s d i p l o i d e xcept 103 ( A n ) . Seed parent H. saccata 136 13'*-3 1 3 5 - 2 105 121 1 3 3 - 1 1 3 3 - 2 1 3 3 - 3 1 3 5 - 1 10? 102b-l 134-2 Pollen parent open p o l l i -nations No. of seed counted with embryos 195 H. saceata ^nixed p o l l e n I, \ tnent H. d i l a t a t a 112a-3 122 mixed pollen 110-3 mixed pollen H. hyperbo-rea mixed p o l l e n 1 0 3 - 3 1 0 3 - 6 emasculated open.without cover very stored •rood d e v e l o p m e n t pollen us£d, slow flevelop-22? very good development no sped, s t a l k broken good A f t e r embryos 424 slow de looking f e r t i l with-out 528 t o t a l f e r t i l i -ty 723 27 Remarks open p o l l i n a t i o n s only c h a f f 27 % mean 403 630 24 development days l a r g e , w e l l 248 eloping, embryos 672 but norm za t i o n a f t e r 18 days i n t r a s p e c i f i c l a r ~ e w ell developed embryos i n s e c t i o n s no seed, spike broken 31 formed i n t e r s p e c i f i c . s m all capsules with very l i t t l e good seed ( s t a l k broken,very slow development no seed, a l l ova-r i e s s e c t i o n e d a l l o v a r i e s sec-t ioned 31 % mean 63 6 . 6 i n t e r s p e c i f i c w e l l developed '.' lar g e embryos a l l o v a r i e s sec-t ioned s t a l k broken 63 ~/° mean c o n t r o l nlant VI. GENERAL CONCLUSIONS. As i n d i c a t e d i n "M a t e r i a l s and Methods" e a r l i e r , r e c i p r o c a l crosses between H. d i l a t a t a , H. hyperborea and H. saccata were made. Besides the i n t e r s p e c i f i c p o l l i n a t i o n s s e v e r a l i n t r a s p e c i f i c crosses w i t h i n each of the three species seemeida-indicated and v a r i o u s c o n t r o l s were set up. The r e s u l t s of crosses and c o n t r o l s have been discussed i n the previous chapter. I t remains t o point out some conclusions which seemed t o a r i s e from the present study. A l l p o l l i n a t e d f l o r e t s produced seed pods. Some capsules be-came very l a r g e , e s p e c i a l l y the t e t r a p l o i d ones, whereas the d i p l o i d H. hyper- borea p l a n t s had the smallest capsules and the l e a s t seed. A great deal of s seed seemed t o r i p e n , a l l mature ovaries having produced some seed w i t h w e l l formed embryos. Empty seeds were found mostly a f t e r i n t e r s p e c i f i c crosses and were foreshadowed by e a r l y breakdown i n the c e l l s of the embryo sac, n o t i c e a b l e from the 2 - 4 c e l l e d stage of the female gametophyte ( F i g . 1 3 -1 , 2 , 3 ) onward, a f t e r a r r i v a l of the f o r e i g n p o l l e n tube i n the ovary. No d i f f e r e n c e could be detected i n ovule and embryo development among the three species studied, whether p o l l i n a t e d n a t u r a l l y or a r t i f i -c i a l l y . A comparison of ovule and embryo development i n p l a n t s c o l l e c t e d from the w i l d w i t h development a f t e r i n t e r s p e c i f i c p o l l i n a t i o n s gave no i n d i c a t i o n t h a t hybrids develop i n a d i f f e r e n t way from species, except f o r an increase i n the amount of breakdown. In i n t r a s p e c i f i c crosses development from p o l l i n a t i o n to f e r t i l i -z a t i o n and from f e r t i l i z a t i o n t o m a t u r a t i o n o f the embryo i s r a p i d . I t i s s l o w e r a f t e r i n t e r s p e c i f i c c r o s s i n g s where, presumably, e f f e c -t i v e i s o l a t i n g mechanisms f u n c t i o n . The 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 the s p e c i e s s t u d i e d are s l i g h t , as are t h e i r h a b i t a t p r e f e r e n c e s ; H. d i l a t a t a seems to p r e f e r more open a r e a s , o f t e n i n f u l l sun, whereas the two o t h e r s p e c i e s seem to seek out the p r o t e c t i o n g i v e n by o t h e r v e g e t a t i o n . Comparing my c r o s s e s w i t h t h o s e made by Swamy (1948) on 9 I n d i a n H a b e n a r i a s p e c i e s , w i t h the work of Heusser (1914) on H i r -cinum himantoglossum and w i t h t h a t of C o c u c c i (1961) on a s o u t h -American o r c h i d , a l l b e l o n g i n g t o the t r i b e O r c h i d e a e , a g a i n I f i n d s i m i l a r i t i e s r a t h e r than d i f f e r e n c e s . However, t h e r e i s a s l i g h t d i f f e r e n c e between t e r r e s t r i a l o r c h i d s and e p i p h y t e s i n o v u l e i n i t i a a fcion:,-, which i n the t e r r e s t r i a l h a b e n a r i a s t a k e s p l a c e b e f o r e a r r i v a l o f the p o l l e n , i n e p i p h y t i c ones a f t e r a r r i v a l of the p o l l e n . That gene f l o w e x i s t s and i s i n o p e r a t i o n among the t h r e e H a b e n a r i a s p e c i e s i s p r o b a b l e as i n d i c a t e d by the s u c c e s s of the a r t i f i c i a l p o l l i n a t i o n s . The c r o s s e s seem to suggest t h a t t h e r e i s c l o s e r r e l a t i o n s h i p between H. hyperborea and H. s a c c a t a than between e i t h e r o f th e s e and H. d i l a t a t a . That the s p e c i e s are a b l e t o r e t a i n t h e i r d i s c r e e t n e s s i n s p i t e of t h e i r a b i l i t y t o c r o s s can be e x p l a i n e d by e f f e c t i v e i s o l a t i n g mechanisms which are broken o n l y o c c a s i o n a l l y . As a whole the t h r e e s p e c i e s s t u d i e d ' f o l l o w c l o s e l y the c o u r s e of development found a l s o i n o t h e r members o f t h e i r t r i b e . V I I . SUMMARY. H. d i l a t a t a , H. hyperborea and H. s a c c a t a were c r o s s e d r e c i p r o -c a l l y and i n t r a s p e c i f i c c r o s s e s were made w i t h i n each s p e c i e s . C o n t r o l s were s e t up to t e s t the t e c h n i q u e used, the presence of p o l l i n a t o r s on the r o o f and the b r e e d i n g system i n o p e r a t i o n i n the t h r e e s p e c i e s . H. d i l a t a t a and H. hyperborea can be o u t - c r o s s i n g o r autogamous, depending on the e a r l y or l a t e a r r i v a l o f the i n s e c t s . H. s a c c a t a i s s e l f - i n c o m p a t i b l e . Development o f the embryos was s t u d i e d a f t e r i n t r a - and i n t e r s p e c i -f i c c r o s s e s had been made. I t was found t h a t embryos o f both s p e c i e s and h y b r i d s show the same development s t a g e s , except f o r l o n g e r p e r i o d s of time from p o l l i n a t i o n to f e r t i l i z a t i o n and from f e r t i l i z a t i o n to the m a t u r a t i o n o f the embryos i n h y b r i d o f f s p r i n g . Comparing the development o f a r t i f i c i a l l y p o l l i n a t e d p l a n t s to those c o l l e c t e d i n the w i l d , I found o n l y s i m i l a r i t i e s . Seeds of mature o v a r i e s o f both i n t r a - and i n t e r s p e c i f i c c r o s s e s were counted and a mean v a l u e o f f e r t i l i t y e s t a b l i s h e d . As e x p e c t e d , the i n t r a s p e c i f i c c r o s s e s y i e l d e d l a r g e amounts o f seed w i t h s e e m i n g l y h e a l t h y , w e l l formed embryos. I n t e r s p e c i f i c c r o s s e s gave b e t t e r r e s u l t s when H. hyperborea was c r o s s e s w i t h H. s a c c a t a . i n d i c a t i n g c l o s e r r e l a t i o n s h i p , then when H. d i l a t a t a was c r o s s e s w i t h e i t h e r o f the two o t h e r s p e c i e s . 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