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Genotypic and phenotypic variation in populations of Daphnia pulex Krepp, Susan Rose 1977

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GENOTYPIC AND PHENOTYPIC VARIATION IN POPULATIONS OF DAP HNIA PULEX BY SUSAN ROSE KREPP B.Sc., The P e n n s y l v a n i a S t a t e U n i v e r s i t y , 1974 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department of Zool o g y We a c c e p t t h i s t h e s i s as con f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA SEPTEMBER, 1977 COPYRIGHT SUSAN ROSE KREPP, 1977 In present ing th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f r ee ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by h is representa t ives . It is understood that copying or p u b l i c a t i o n of th is thes is for f i n a n c i a l gain sha l l not be allowed without my wr i t ten permission. Department of Zoology  The Un ivers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1WS D a t e September 29. 1977 i Abstract Because of t h e i r reproductive pattern, parthenogenetic organisms may have limited genetic v a r i a t i o n and may rel y on alternative mechanisms other than genetic d i v e r s i t y f o r maintaining phenotypic v a r i a b i l i t y and adapting to the environment. This hypothesis was tested by measuring genotypic and phenotypic variation in several populations of Daphnja jaulex , an apomictic, parthenogenetic cladoceran. Genotypic variation measured by starch gel electrophoresis indicated 0% variable l o c i in 3 species of Da£hnia i n the lower mainland around Vancouver, B.C. and 3855 polymorphic l o c i i n Near Roundup, a pond in the Interi o r of the province near Williams Lake. Differences in environmental conditions and electrophoretic patterns provide a rat i o n a l e f o r comparing phenotypic v a r i a t i o n i n 3 e l e c t r o p h o r e t i c a l l y , p h y s i c a l l y and geographically s i m i l a r ponds, P2, P4, and P5, and i n an ele c t r o p h o r e t i c a l l y polymorphic population (NR) which are physically and geographically d i s t i n c t . Means and variances of 5 morphological and 1 to 6 reproductive characters were compared within and among clones i n each population and among populations and indicated the following: 1) There were s i g n i f i c a n t differences i n means for most characters among clones and among populations regardless of electrophoretic s i m i l a r i t y or d i s s i m i l a r i t y among clones or populations, 2) There was greater i n t r a c l o n a l v a r i a t i o n than i n t e r c l o n a l v a r i a t i o n i n a l l p o p u l a t i o n s f o r a l l c h a r a c t e r s , 3) t h e r e was s i g n i f i c a n t l y g r e a t e r t o t a l v a r i a n c e , i n t r a c l o n a l v a r i a n c e , and i n t e r c l o n a l v a r i a n c e i n P2 t h a n i n HR, and 4) v a r i a n c e s were p a r t i t i o n e d e q u a l l y w i t h i n and among c l o n e s i n P2 whereas th e g r e a t e s t % v a r i a t i o n i n NB was w i t h i n c l o n e s . These d a t a s uggest an i n v e r s e r e l a t i o n o f g e n e t i c and p h e n e t i c v a r i a b i l i t y i n t h e s e p o p u l a t i o n s o f Daphnia and suggest t h a t P2 and NR are examples of a d a p t a t i o n by i n d i v i d u a l and p o p u l a t i o n a l h o m e o s t a s i s . P2 i n d i v i d u a l s which are e l e c t r o p h o r e t i c a l l y monomorphic may r e l y on extreme p h e n o t y p i c p l a s t i c i t y i n o r d e r t o adapt t o the environment. NS Daphnia may a l s o r e l y on p h e n o t y p i c p l a s t i c i t y t o a l e s s e r e x t e n t as demonstrated by t h e l a r g e % v a r i a t i o n w i t h i n c l o n e s , however, t h e r e l a t i v e l y s m a l l a b s o l u t e v a r i a n c e and t h e e l e c t r o p h o r e t i c v a r i a t i o n may i n d i c a t e a d a p t a t i o n by g e n e t i c changes i n t h e p o p u l a t i o n . These p o s s i b l e s t r a t e g i e s have been f u r t h e r i n t e r p r e t e d r e l a t i v e t o s e l e c t i o n and t e m p o r a l s t a b i l i t y o f the environment. P h e n o t y p i c p l a s t i c i t y and l a c k o f e l e c t r o p h o r e t i c v a r i a t i o n i n Daphnia and i n o t h e r p a r t h e n o g e n e t i c and i n b r e e d i n g organisms suggest t h a t t h e s e o r g a n i s m s a r e not dependent on g e n e t i c changes i n t h e p o p u l a t i o n t o s u r v i v e . There i s however e v i d e n c e o f g e n e t i c and p h e n e t i c v a r i a t i o n i n p a r t h e n o g e n e t i c organisms comparable t o v a r i a t i o n i n s e x u a l l y r e p r o d u c i n g o r g anisms and t h i s s u g g e s t s t h a t g e n e t i c v a r i a t i o n i s not n e c e s s a r i l y c o n s t r a i n e d by t h e mode o f r e p r o d u c t i o n . i i i T a b l e Of C o n t e n t s L I S T OF FIGURES V L I S T OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v i i ACKNOWLEDGEMENTS .. . . v i i i INTRODUCTION . . . . . . . . . . . . . . . . . . 1 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 RESULTS AND DISCUSSION 19 E l e c t r o p h o r e s i s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Co m p a r i s o n Of P2, P4, And P5; F i e l d Data 25 C o m p a r i s o n Of P2, P4, And P5: Lab And F i e l d Data ....... 34 C o m p a r i s o n Of P2, P4, And P5: Lab Data . . . . . . . . . . . . . . . . . 43 C o m p a r i s o n s Of P2, P4, And P5: I n t e r And I n t r a c l o n a l V a r i a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 C o m p a r i s o n Of P2, P4, And P5; Summary . . . . . . . . . . . . . . . . . . 53 Co m p a r i s o n Of P2 And NR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 C o m p a r i s o n s Of P2 And NS: I n t r a p o p u l a t i o n R e s u l t s F o r P2 Com p a r i s o n s Of P2 And NR: I n t r a p o p u l a t i o n R e s u l t s F o r NR I n t e r p o p u l a t i o n C o m p a r i s o n s Of P2 And NR: Means .. . . . . . . 62 I n t e r p o p u l a t i o n C o m p a r i s o n s Of P2 And NR: V a r i a n c e s .... 63 Co m p a r i s o n Of P2 And NR: Summary 69 C o m p a r i s o n Of P2 And NR: T e m p e r a t u r e E x p e r i m e n t . . . . . . . . 72 FINAL DISCUSSION 81 E n v i r o n m e n t a l S t a b i l i t y Of P2 And NR . . . . . . . . . . . . . . . . . . . . 87 LITERATURE CIT L I S T OF FIGURES F i g u r e 1 : L o c a t i o n Of Ponds I n The Lower m a i n l a n d Near V a n c o u v e r , And I n C e n t r a l B.C. Near W i l l i a m s L a k e . 11 F i g u r e 2 : M o r p h o l o g i c a l M e a s u r e s Of P h e n o t y p i c V a r i a t i o n . 16 F i g u r e 3 : E l e c t r o p h o r e t i c P o l y m o r p h i s m At The AKP, ES, And X* A P Xi oo x* .x • * • « • • » •••••21 F i g u r e 4 : D i s t r i b u t i o n Of Body L e n g t h s In F i e l d And L a b P o p u l a t i o n s Of D a p h n i a p u l e x From P2. . . . . . . . . . . . . . . . . . . 28 F i g u r e 5 : D i s t r i b u t i o n Of Body L e n g t h s I n F i e l d And Lab P o p u l a t i o n s Of D a p h n i a p u l e x From P5. ..................30 F i g u r e 6 : D i s t r i b u t i o n Of Body L e n g t h I n F i e l d And L a b P o p u l a t i o n s From P5. ... ...................32 F i g u r e 7 : Means And 95% C o n f i d e n c e L i m i t s F o r Body L e n g t h In F i e l d And L a b P o p u l a t i o n s O f D a g h n i a p u l e x From P2, P4, And P5. .............. v.............................38 F i g u r e 8 : Means And 95% C o n f i d e n c e L i m i t s F o r Egg Number I n F i e l d And Lab P o p u l a t i o n s Of P2, P4, And P5. ......... 40 F i g u r e 9 : Means And 95% C o n f i d e n c e L i m i t s F o r The T h r e e P r i n c i p a l Component V a r i a b l e s Of P2, P4, And P5. .......47 F i g u r e 10 : Means And 95% c o n f i d e n c e L i m i t s F o r Body L e n g t h I n P2 And NR D a p h n i a R e a r e d At T h r e e T e m p e r a t u r e s . .....77 F i g u r e 11 : Means And 95% C o n f i d e n c e L i m i t s F o r Egg Number I n P2 And NR D a p h n i a R e a r e d At T h r e e T e m p e r a t u r e s . .....79 v i L I S T OF TABLES T a b l e 1. E l e c t r o p h o r e t i c V a r i a t i o n In P a r t h e n o g e n e t i c And I n b r e e d i n g O r g a n i s m s R e p o r t e d I n The L i t e r a t u r e 4 T a b l e 2. Summary O f The A v a i l a b l e E n v i r o n m e n t a l D a t a F o r The L a k e s I n T h i s S t u d y 13 T a b l e 3. Summary Of T h e P h y s i c a l And C h e m i c a l Data F o r T h e P e t e r s o n And Near Roundup Ponds 14 T a b l e 4. Per Cen t a o n o m o r p h i c L o c i I n 22 P o p u l a t i o n s Of Da.2hn.ia joulex , DaBknia. £ o s e a , And D a p h n i a l a e v i s .....20 T a b l e 5. N u m e r i c a l D e s i g n a t i o n s F o r A l l e l e s M e asured From The O r i g i n , And A l l e l e F r e q u e n c i e s F o r T h r e e S p e c i e s Of Dap h n i a . . . . . . . . . . . . . . .....24 T a b l e 6 . E s t i m a t e s Of The Mean, V a r i a n c e , And 95% C o n f i d e n c e L i m i t s F o r P2, P4, And P5 F i e l d P o p u l a t i o n s .26 T a b l e 7. C o m p a r i s o n Of L a b And F i e l d V a r i a n c e s I n P2, P4, And' P5 ......... . .......35 T a b l e 8. C o m p a r i s o n Of Lab And F i e l d V a r i a n c e s {% V a r i a t i o n I n P2, P4, And P5 ,...36 T a b l e 9. E s t i m a t e s Of The Mean, V a r i a n c e , And 95% C o n f i d e n c e L i m i t s F o r P2, P4, And P5 Lab P o p u l a t i o n s «..42 T a b l e 10. C o m p a r i s o n Of W i t h i n And Among C l o n e V a r i a t i o n I n P2, P4, And P5 50 T a b l e 11. C o m p a r i s o n Of V a r i a t i o n W i t h i n And Among C l o n e s And Among P o p u l a t i o n s P2, P4, And P5 ,...52 T a b l e 12. Summary Of The R e s u l t s From C o m p a r i s o n s Of P2, P4, And P5 .54 v i i T a b l e 13. E s t i m a t e s Of Means, V a r i a n c e s , And 951 C o n f i d e n c e L i m i t s For P2 And NS .58 T a b l e 14. Comparison Of W i t h i n And Among Clone V a r i a t i o n I n P2 And NE 59 T a b l e 15. R a t i o Of V a r i a n c e s W i t h i n And Among C l o n e s I n P2 And NR From Untransformed And L o g a r i t h m i c a l l y Transformed Data .......,..................... ..........60 T a b l e 16. Comparison Of V a r i a n c e s For NR And P2 ...........64 T a b l e 17. F T e s t s Comparing R e l a t i v e V a r i a n c e s From Transformed Data W i t h i n And Among C l o n e s Between P o p u l a t i o n s P2 And NS ..................................66 T a b l e 18. Comparison Of V a r i a t i o n W i t h i n And Among C l o n e s And Between P o p u l a t i o n s P2 And NR 63 T a b l e 19. Summary Of The G e n e t i c Data From P2 And NS ......70 T a b l e 20. Means And V a r i a n c e s For M o r p h o l o g i c a l And R e p r o d u c t i v e C h a r a c t e r s P2 And NR Reared At Three Temperatures ...........................................73 T a b l e 21. Comparison Of Means And V a r i a n c e s I n P2 And NR At Three Temperatures ................. . . . . . . . . , . . . . , 7 6 T a b l e 22. Comparisons Of M o r p h o l o g i c a l C h a r a c t e r s I n P2 And NR . . ,. , . . 85 Tab l e 23. Means And V a r i a n c e s For F i t n e s s Based On Number Of S u r v i v o r s And Number Of Eggs I n P2 And NR ...........90 v i i i ACKNOWLEDGEMENTS W i t h o u t t h e a d v i c e , s u p p o r t , and c r i t i c a l e v a l u a t i o n o f Dr. W i l l i a m E. N e i l l t h i s s t u d y would n o t h a v e been p o s s i b l e . He has been c o m p l e t e l y u n s e l f i s h w i t h h i s t i m e and e n e r g y and I am i n d e b t e d t o him f o r h i s g e n u i n e i n t e r e s t i n my d e v e l o p m e n t a s a g r a d u a t e s t u d e n t and r e s e a r c h e r . I am a l s o i n d e b t e d t o Mark Denny f o r h i s u n e n d i n g p a t i e n c e and c o n f i d e n c e i n me t h r o u g h o u t t h i s s t u d y , and t o my p a r e n t s and t h e t w i n s who have u n g u e s t i o n i n g l y s u p p o r t e d and e n c o u r a g e d me i n my i n t e r e s t i n b i o l o g y . The m o t i v a t i o n and f r i e n d s h i p o f B i l l C l a r k , Judy Myers, J u d y N e i l l , A d r i e n n e P e a c o c k , John Spence, and C a r l W h i t n e y have b e e n i n v a l u a b l e t o me t h r o u g h o u t t h i s s t u d y . 1 IHTBODUCTION "The b a s i s o f i n d i v i d u a l i t y i s v a r i a t i o n . V a r i a t i o n i s t h e m a t e r i a l o f s c i e n c e and v a r i a t i o n among t h e members o f a s p e c i e s i s t h e m a t e r i a l o f g e n e t i c s " ( C l a r k e , 197 4) . C l a s s i c a l l y p h e n o t y p i c v a r i a t i o n h as been measured as m o r p h o l o g i c a l and p h y s i o l o g i c a l v a r i a b i l i t y w i t h i n and among p o p u l a t i o n s . P h e n o t y p i c v a r i a t i o n h as been s t u d i e d i n a l a r g e number o f s p e c i e s and f o r a few s p e c i e s p o l y m o r p h i s m s o f some c h a r a c t e r s , s u c h a s s h e l l c o l o r i n s n a i l s a n d wing c o l o r i n moths, have been d e m o n s t r a t e d t o be s e l e c t i v e l y i m p o r t a n t ( F o r d , 1976) . G e n e t i c v a r i a t i o n has been d e t e r m i n e d by measurements o f q u a l i t a t i v e and q u a n t i t a t i v e v a r i a t i o n i n c h a r a c t e r s whose g e n e t i c b a s i s i s known, and by chromosomal v a r i a b i l i t y among p o p u l a t i o n s o r s p e c i e s . Hore r e c e n t l y g e n e t i c v a r i a t i o n h a s been measured by enzyme p o l y m o r p h i s m s d e t e c t e d by e l e c t r o p h o r e s i s and t h e d e g r e e o f e l e c t r o p h o r e t i c v a r i a t i o n o f p r o t e i n i n b o t h v e r t e b r a t e s and i n v e r t e b r a t e s i s c o n s i d e r a b l e ( S e l a n d e r , 1976). The m a i n t e n a n c e o f t h i s v a r i a t i o n has been i n t e r p r e t e d by a number o f e x p l a n a t i o n s b a s e d on s e l e c t i o n ( L e v i n s , 1968); t h e o r g a n i s m s p e r c e p t i o n o f t h e e n v i r o n m e n t a s b e i n g c o a r s e - o r f i n e - g r a i n e d a s s o c i a t e d w i t h t h e m o b i l i t y and t h e h o m e o s t a s i s o f t h e o r g a n i s m , ( S e l a n d e r and Kaufman, 1973); r e p r o d u c t i v e s t r a t e g i e s ; r a t e o f m u t a t i o n (Crow and K i m u r a , 1965) and r a t e o f gene f l o w . T h i s s t u d y d e a l s w i t h g e n o t y p i c and p h e n o t y p i c 2 v a r i a t i o n a s s o c i a t e d w i t h a u n i q u e r e p r o d u c t i v e s t r a t e g y , p a r t h e n o g e n e s i s . P h e n o t y p i c v a r i a t i o n i s c l a s s i f i e d by measures o f m o r p h o l o g i c a l and p h y s i o l o g i c a l v a r i a b i l i t y and g e n o t y p i c v a r i a t i o n i s measured by e l e c t r o p h o r e s i s and by c o m p a r i s o n s o f i n t e r - and i n t r a c l o n a l v a r i a t i o n . One might e x p e c t l e s s v a r i a t i o n i n p a r t h e n o g e n s o r i n a s e x u a l l y r e p r o d u c i n g o r g a n i s m s t h a n i n s e x u a l l y r e p r o d u c i n g o r g a n i s m s due t o 1) l a c k o f random a s s o r t m e n t and r e c o m b i n a t i o n o f chromosomes, and 2) d i r e c t i o n a l s e l e c t i o n e l i m i n a t i n g most g e n o t y p e s . , D a t a b o t h s u p p o r t i n g and c o n t r a d i c t i n g t h i s h y p o t h e s i s h a v e been r e p o r t e d f o r b o t h m o r p h o l o g i c a l and f o r e l e c t r o p h o r e t i c v a r i a b i l i t y . Reduced p h e n o t y p i c v a r i a t i o n has been d e m o n s t r a t e d i n p a r t h e n o g e n e t i c D a p h n i a l o n g i s p i n a and i n a n o t h e r c l a d o c e r a n , H o i n a spp i n c o m p a r i s o n s o f s e x u a l l y r e p r o d u c e d and p a r t h e n o g e n e t i c a l l y r e p r o d u c e d i n d i v i d u a l s f r o m t h e same c l o n e s ( B a n t a , 1939). B a n t a and Wood i n an e a r l i e r s t u d y , however, r e p o r t e d s i m i l a r amounts o f p h e n o t y p i c v a r i a t i o n i n D a p h n i a r e p r o d u c e d s e x u a l l y and a s e x u a l l y {1927). O t h e r a p o m i c t i c o r g a n i s m s s u c h a s d a n d e l i o n s ( S o l b r i g , 1971), l i z a r d s ( W r i g h t and Lowe, 1967) and w e e v i l s ( S u o m a l a i n e n , 196 9) have a l s o been shown t o be e x t r e m e l y v a r i a b l e . A t o t a l l a c k o f e l e c t r o p h o r e t i c v a r i a t i o n has been o b s e r v e d i n t h r e e s p e c i e s o f bees which a r e h a p l o - d i p l o i d ( S n y d e r , 1974), i n Sumina d e c o l l a t a a E u r o p e a n l a n d s n a i l w h i c h i s a f a c u l t a t i v e s e l f e r ( S e l a n d e r and Kaufman, 1973 ) , and i n a t r i p l o i d l i z a r d , C n e m i d o p h o r u s t e s s e l a t u s which r e p r o d u c e s p a r t h e n o g e n e t i c a l l y ( P a r k e r and S e l a n d e r , 1976).. On t h e o t h e r hand, l a r g e amounts o f e l e c t r o p h o r e t i c v a r i a t i o n h a v e been r e p o r t e d f o r p a r t h e n o g e n e t i c 3 p o p u l a t i o n s o f l i z a r d s ( P a r k e r and S e l a n d e r , 1 9 7 6 ) , w e e v i l s ( S u o m a l a i n e n and S a u r a , 1973), c l a d o c e r a n s ( H e b e r t , 1974; Young, u n p u b l i s h e d d a t a ; and S m i t h and F r a s e r , 1976), and i n s e l f - p o l l i n a t i n g w i l d o a t s ( M a r s h a l l and a i l a r d , 1970). D a t a f r o m t h e l i t e r a t u r e on e l e c t r o p h o r e t i c v a r i a t i o n i n p a r t h e n o g e n e t i c a l l y r e p r o d u c i n g o r g a n i s m s a r e summarized i n T a b l e 1. P r o p o s e d e x p l a n a t i o n s f o r m a i n t e n a n c e o f v a r i a t i o n i n p a r t h e n o g e n s i n c l u d e i n c r e a s e d i n c o r p o r a t i o n o f m u t a t i o n s , s t a b i l i z i n g s e l e c t i o n , h e t e r o s i s , and l a r g e amounts o f i m m i g r a t i o n . D a p h n i a r e p r o d u c e p a r t h e n o g e n e t i c a l l y d u r i n g l a r g e p a r t s o f t h e y e a r . P a r t h e n o g e n e s i s i n D a p h n i a i s t h o u g h t t o be a m e i o t i c b a s e d on' c y t o l o g i c a l ( M o r t i m e r , 1936) and e l e c t r o p h o r e t i c s t u d i e s ( H e b e r t and H a r d , 1974), e l i m i n a t i n g any v a r i a t i o n due t o r e c o m b i n a t i o n i n t h e o f f s p r i n g . B a c c i , e t a l . (1961 and 1965), however, a r g u e t h a t p a r t h e n o g e n e s i s i s e n d o r a e i o t i c and t h e r e f o r e assume t h a t r e c o m b i n a t i o n c a n g i v e r i s e t o g e n e t i c v a r i a b i l i t y w i t h i n s i n g l e p a r t h e n o g e n e t i c l i n e s o f D a p h n i a -.. The g e n e t i c s i m i l a r i t y o f s i b l i n g s i n t h i s s t u d y i s r e c o g n i z e d by e l e c t r o p h o r e t i c s i m i l a r i t y and, s i n c e no v a r i a t i o n was o b s e r v e d among s i b s e l e c t r o p h o r e t i c a l l y , I have assumed e n d o m e i o s i s i s n o t o c c u r i n g i n t h e s e o r g a n i s m s . D a p h n i a a r e a l s o c a p a b l e o f p r o d u c i n g m ales and s u b s e q u e n t s e x u a l r e p r o d u c t i o n i n r e s p o n s e t o e n v i r o n m e n t a l a n d / o r d e m o g r a p h i c s t i m u l i a s s o c i a t e d w i t h d e c r e a s i n g l i g h t , t e m p e r a t u r e , o r f o o d , and i n c r e a s i n g p o p u l a t i o n d e n s i t y ( S t r o s s , 1969). F e m a l e s u s u a l l y p r o d u c e two e p h i p p i a l e ggs which Table 1 : E l e c t r o p h o r e t i c v a r i a t i o n i n parthenogenetic and inbreeding organisms reported i n the l i t e r a t u r e . organism mode of reproduction v a r i a b l e l o c i t o t a l l o c i reference Rumina d e c o l l a t a Augochiora pura  Lasioglossum zephyrum  Bombus americanorum Drosophila mercatorum Otiorrhynchus scaber (3N) 0. scaber (4N) 0. s i n g u l a r i s (3N) Strophosomus melanogrammus (3N) Cnemidophorus tesselatus (2N) C. tesselatus (3N) f a c u l t a t i v e s e l f - f e r t i l i z a t i o n haplo-diploidy parthenogenesis parthenogenesis parthenogenesis P o e c i l i o p s i s 2 monacha-lucida gynogenesis 0/25 (0%) 0/13 (0%) 0/24 (0%) 0/12 (0%) 5/10 (50%) males 7/12 (58%) females 16/26 (62%) 16/26 (62%) 16/23 (70%) 9/20 (45%) 6/21 (29%) 0/21 (0%) 4/23 (17%) Avena barbata Avena fatua autogamous s e l f -f e r t i l i z a t i o n 5/16 7/13 (31%) (54%) Selander and Kaufman (1973) Snyder (1974) Templeton, Carson, and Sing (1976) Suomalainen and Saura (1973) Parker and Selander (1976) Vrijenhoek and L e s l i e (197 ) Marshall and A l l a r d (1970) Table 1 : Electrophoretic variation in parthenogenetic and inbreeding organisms reported in the literature (cont.). organism mode of variable l o c i reference reproduction total l o c i (%) Simocephalus serrulatus parthenogenesis 5/16 to 9/16 Smith and Fraser (31% to 56%) (1976) Daphnia magna  D. pulex parthenogenesis 4/13 (31%) 3/8 (37%) Hebert (1974) Young (unpub1. data) 6 o v e r w i n t e r i n t h e l a k e and h a t c h a f t e r a p p r o p r i a t e e n v i r o n m e n t a l s t i m u l u s . T h i s r e p r o d u c t i v e s t r a t e g y i d e a l l y c o v e r s a l l b a s e s : i n d i v i d u a l s a r e not o n l y c a p a b l e o f h i g h f e c u n d i t y and r a p i d c o l o n i z a t i o n a s s o c i a t e d w i t h p a r t h e n o g e n e s i s , b u t a r e a l s o c a p a b l e o f d i s p e r s a l o f t h e e g g s , and o f r e o r g a n i z a t i o n o f g e n e t i c m a t e r i a l by s e x u a l r e p r o d u c t i o n . B e c a u s e t h e y a r e a c y c l i c a l p a r t h e n o g e n s , D a p h n i a a r e i n t e r e s t i n g o r g a n i s m s f o r measurement and e v a l u a t i o n o f t h e i m p o r t a n c e o f g e n o t y p i c and p h e n o t y p i c v a r i a t i o n . P h e n o t y p i c and g e n o t y p i c v a r i a t i o n have p r e v i o u s l y been d e s c r i b e d f o r p o p u l a t i o n s o f D a p h n i a ., L a r g e amounts o f p h e n o t y p i c v a r i a t i o n i n D a p h n i a have been d e s c r i b e d among p o p u l a t i o n s i n B r i t i s h C o l u m b i a ( C a r l , 1940) and i n H o r t h A m e r i c a ( B r o o k s , 1957). B e c a u s e o f t h e s e r e g i o n a l d i f f e r e n c e s i n p h e n o t y p e , a c c u r a t e t a x o n o m i c c h a r a c t e r i z a t i o n s o f s p e c i e s has been d i f f i c u l t . P h e n o t y p i c v a r i a t i o n i n D a p h n i a may a l s o be c y c l o m o r p h i c , i n t h a t head and c a r a p a c e m o rphology change t h r o u g h s u c c e s s i v e g e n e r a t i o n s o f p a r t h e n o g e n e t i c f e m a l e s . C y c l o m o r p h o s i s r e s u l t i n g i n c h a n g e s i n e y e d i a m e t e r and l e n g t h o f t a i l s p i n e i s t h o u g h t t o be an a d a p t a t i o n t o p r e d a t o r a v o i d a n c e ( J a c o b s , 1966; Z a r e t , 1972; Dodson, 1974) and i s presumed t o be i n d u c e d by i n c r e a s i n g t e m p e r a t u r e and c o r r e l a t e d w i t h f o o d s u p p l y and t u r b u l e n c e o f t h e e n v i r o n m e n t ( B r o o k s , 1946; r e v i e w by H u t c h i n s o n , 1967). E l e c t r o p h o r e t i c measures o f g e n e t i c v a r i a t i o n i n c l a d o c e r a n s a r e c o m p a r a b l e t o t h o s e o f s e x u a l l y r e p r o d u c i n g s p e c i e s . H a r r i s (1966) and L e w o n t i n and Hubby (1966) i n s t u d i e s 7 of humans and D r o s o p h i l a concurred i n f i n d i n g approximately 30% v a r i a b l e l o c i (comparable t o v a r i a t i o n i n Daphnia magna and Paphnia pulex which are v a r i a b l e at 31 t o 38% of a l l l o c i ) . Simocephalus a l s o was h i g h l y polymorphic with 33 to 60 % v a r i a b l e l o c i i n s e v e r a l p o p u l a t i o n s (Smith and F r a s e r , 1976). Phenotypic v a r i a t i o n i s presumably i n f l u e n c e d by the genotype of the i n d i v i d u a l and by the environment i n which the organism l i v e s , and i n t h i s study a model r e l a t i n g genotype, phenotype, and environment i n Daphnia w i l l be proposed.,Falconer (1965) proposed an a d d i t i v e model of v a r i a n c e s i n which phenotypic v a r i a t i o n i s the sum of g e n e t i c and environmental v a r i a t i o n . F u r t h e r , one would expect some i n t e r a c t i o n o f the genotype and the environment i n d e s c r i b i n g mean phenotype i n a po p u l a t i o n . T h i s i n t e r a c t i o n of genotypic and phenotypic v a r i a t i o n can be d i s c u s s e d i n terms of two a l t e r n a t i v e s t r a t e g i e s a s s o c i a t e d with i n d i v i d u a l and p o p u l a t i o n a l homeostasis (Thoday, 1953; Lewontin, 1957, and L e v i n s , 1965). P o p u l a t i o n s may adapt t o a v a r i e t y of environments by i n d i v i d u a l f l e x i b i l i t y i n which each i n d i v i d u a l i s capable of modifying the expression of the genotype i n response to the environment. Phenotypic p l a s t i c i t y , the amount by which the e x p r e s s i o n of the c h a r a c t e r i s t i c of a genotype i s changed by d i f f e r e n t environments (Bradshaw, 1965) , can permit a s i n g l e genotype t o assume d i f f e r e n t phenotypes. T h i s i s p a r t i c u l a r l y advantageous i n a c c l i m a t i n g t o changes i n the environment which are of s h o r t e r d u r a t i o n than the g e n e r a t i o n time of the organism. P o p u l a t i o n s may a l s o adapt t o a v a r i e t y o f environments by using the d i f f e r e n t i a l f i t n e s s of the i n d i v i d u a l s where m u l t i p l e 8 g e n o t y p e s a r e e a c h a d a p t e d t o a s p e c i f i c e n v i r o n m e n t . P h e n o t y p i c p l a s t i c i t y i n t h e s e p o p u l a t i o n s c a n e n a b l e d i f f e r e n t g e n o t y p e s t o assume a s i n g l e p h e n o t y p e . L e v i n s (1965) p r o v i d e s a m a t h e m a t i c a l model o f t h e s e s t r a t e g i e s and s u g g e s t s an i n v e r s e r e l a t i o n s h i p o f g e n o t y p i c t o p h e n o t y p i c v a r i a t i o n . D i f f e r e n c e s i n t h e p o p u l a t i o n s t r u c t u r e o f Avena b a r b a t a . t h e s l e n d e r w i l d o a t , and ft. f a t u a , t h e common w i l d o a t , have been e x p l a i n e d by t h e s e two s t r a t e g i e s ( J a i n and M a r s h a l l , 1 9 6 7 ) ; and a s p r e d i c t e d by L e v i n s , A. b a r b a t a i s g e n e t i c a l l y l e s s v a r i a b l e and p h e n o t y p i c a l l y more v a r i a b l e t h a n A. f a t u a . To d e s c r i b e t h e a d a p t i v e s t r a t e g i e s i n D a p h n i a one n e e d s t o e v a l u a t e t h e d e g r e e o f a d a p t a t i o n , i e . t h e f i t n e s s o f t h e o r g a n i s m , by m e a s u r i n g g e n o t y p i c and p h e n o t y p i c v a r i a t i o n o f e c o l o g i c a l l y i m p o r t a n t c h a r a c t e r s r e l a t i v e t o t h e s t a b i l i t y o f t h e e n v i r o n m e n t . , I n t h i s a n a l y s i s o f v a r i a t i o n a model d e s c r i b i n g t h e r e l a t i o n s h i p o f g e n o t y p e , p h e n o t y p e , and e n v i r o n m e n t i n D a p h n i a £ u l e x p o p u l a t i o n s w i l l be p r o p o s e d and d i s c u s s e d r e l a t i v e t o i n d i v i d u a l and p o p u l a t i o n a l h o m e o s t a s i s . P h e n o t y p i c v a r i a t i o n ( d e t e r m i n e d by means and v a r i a n c e s o f m o r p h o l o g i c a l and p h y s i o l o g i c a l p a r a m e t e r s ) a r e compared i n f i e l d , f i e l d and l a b , and l a b p o p u l a t i o n s from t h r e e l o w e r m a i n l a n d ponds a t 122 39« W, 49 01»N ( d e s i g n a t e d P2, P4, and P 5 ) . P h e n o t y p i c v a r i a t i o n i s a l s o compared f o r l a b p o p u l a t i o n s f r o m two ponds, P2 i n t h e l o w e r m a i n l a n d , and Near Roundup (NR) a t 122 30' W, 52 00» N i n c e n t r a l B r i t i s h C o l u m b i a . The r a t i o n a l e f o r c o m p a r i s o n s o f P2, P4, and P5 a r e b a s e d on t h e 9 e l e c t r o p h o r e t i c s i m i l a r i t y o f t h e p o p u l a t i o n s and t h e p h y s i c a l , and g e o g r a p h i c s i m i l a r i t y o f t h e pondSi R a t i o n a l e f o r c o m p a r i s o n s o f P2 and NR a r e ba s e d on t h e e l e c t r o p h o r e t i c d i s s i m i l a r i t y o f t h e s e p o p u l a t i o n s and t h e e x i s t e n c e o f e n v i r o n m e n t a l d i f f e r e n c e s among ponds., V a r i a t i o n i s compared h i e r a r c h i c a l l y 1) between t h e l o w e r m a i n l a n d and c e n t r a l B.C. R e g i o n s , 2) among p o p u l a t i o n s P2, P4, P5, and N.R., 3) among c l o n e s i n e a c h p o p u l a t i o n and 4) among i n d i v i d u a l s w i t h i n e a c h c l o n e . 10 RATERIALS AND METHODS Da.2hnia were sampled i n 22 ponds, two i n central B r i t i s h Columbia about 35 km west of Williams Lake near Eiske Creek and 20 i n the lower mainland near Vancouver (Fig. 1). Three species, M£hnia fiulex , DaEfeaiS rosea , and Daphnia l a e v i s , were sampled i n 12, 5, and 5 ponds respectively. Several tows from various locations in the pond were taken using a Wisconsin net 30 cm i n mouth diameter with 220 nitex mesh towed from the shore or from a boat. There has been l i t t l e attempt to quantify physical and chemical properties in these ponds although area, depth, vegetation, and s t a b i l i t y of these ponds are reported i n Table 2. This study deals primarily with several ponds in the lower mainland and one i n central B r i t i s h Columbia and further description of these ponds i s given i n Table 3. Daphnia fiulex used i n the lab experiments were chosen a r b i t r a r i l y from f i e l d samples and reared separately i n 40 ml p l a s t i c v i a l s i n a 1:1 d i l u t i o n of pond water and dechlorinated water. Animals were maintained at 15 C and at 16-8 light-dark hours and were fed on every t h i r d day an aguarium (lab) culture of u n i c e l l u l a r algae, primarily C h l o r e l l a , diluted 1:4 with dechlorinated water. Phenotypic variation at the f i r s t reproductive instar was determined by morphological and physiological measurements. Length, width, and head diameter (indicators of body size and shape); and length of t a i l spine and eye diameter (presumed to 11 F i g u r e 1 : L o c a t i o n V a n c o u v e r and W i l l i a m s L a k e . o f ponds i n t h e l o w e r m a i n l a n d n e a r i n t h e I n t e r i o r o f B r i t i s h C o l u m b i a n e a r 12 Table 2 : Summary of the available environmental data for the lakes i n this study. Daphnia sp. Fish Elev. (m) S.A. (ha) depth (m) pH S t a b i l i t y UBC Research Forest * Eunice Placid Gwendoline Katherine UBC Campus Nitobe Gardens Langley Pl-A P2-A P7 Riggs Newhouse PI P2 P3 PA P5 P6 P8 Mcleans UBC Campus Library Bumaby Deer Lake Williams Lake ** NR Box 22 D. rosea 480 cutthroat trout 510 522 505 D. laevis v D. pulex carp trout 30 10 30 100 945 945 18.2 1.6 13.0 20'. 7 <.05 <.01 <.01 36.0 5.06 42 7 27 29 @2 i l < 1 6.4 5.5-6.6 6.6 6.6 6.5-6.7 6.5-6.7 6.4 6.1 6.1 6.0-6.1 6.2- 6.3 6.3- 6.4 6.0 6.8-7.0 8.1-8.6 permanent temporary permanent temporary permanent * Northcote and Clarotto, 1975 ** Topping, 1969 Table 3 : A summary o f the p h y s i c a l c h e m i c a l d a t a f o r the P e t e r s o n ponds and NR. Dates of c o l l e c t i o n are i n d i c a t e d i n parentheses from 1976. Daphnia sp. P P m temp. (C) umho P1A/P2A l a e v i s (4/26) 13.2 (4/26) 14-17 (5/10) 50 P2 p u l e x (5/19) (6/18). (6/23) 0.6 0.8 1.4 (5/19) (6/8) (6/23) 7-7.5 10 9 (5/10) 35 P3 p u l e x (4/26) (5/19) 9.2 8.1 1.4 (4/26) (5/19) 16 17 (5/10) 38 P4 p u l e x (4/26) (5/19) (6/8) (6/23) 1.4 1.2 1.4 2.8 (4/26) (5/19) (6/8) (6/23) 10-10.5 7.5 9 9 (5/10) . 38 P5 p u l e x (4/26) (5/19) 1.3 1.2 (4/26) (5/19) (6/8) 10.5 8 11 (5/10) 35 P7 l a e v i s (4/26) 4.5 (4/26) (5/19) 12-15 11.5-15 (5/10) 30 P8 p u l e x (5/19) (6/8) (6/23) 4.7 3.6 2.2 (5/19) (6/8) (6/23) 7.5 9 9-9.5 P I p u l e x (6/8) (6/23) 0.9 1.2 (6/8) (6/23) 11 9.5 NR * p u l e x (5/12/66) (7/27/66) 4.4 1.2 (5/12/66) (7/26/66) 14.4 16.4-18.9 (5/12/66) (7/27/66) 1, 182 1,485 _PJL (4/26) (4/26) (5/10) (4/26) (5/10) (4/26) (5/10) (4/26) .(5/10) (5/10) 6.1 6.1' 6.1 6.1 6.1 6.0 6.1 6.4 6.4 6.4 (5/12/66) 8.6 (7/27/66) 8.1 * NR da t a from Toppings, 1969 15 be e c o l o g i c a l l y i m p o r t a n t w i t h r e g a r d s t o p r e d a t o r a v o i d a n c e ( Z a r e t , 1972; Dodson, 1974) were measured w i t h an o c u l a r m i c r o m e t e r a t 50x m a g n i f i c a t i o n on a W i l d d i s s e c t i n g m i c r o s c o p e and a r e r e c o r d e d a s m i c r o n s i n t h e t e x t ( F i g . 2 ) . The number o f e g g s a t t h e p r i m a p a r o u s i n s t a r was t h e p r i m a r y measure o f p h y s i o l o g i c a l v a r i a b i l i t y , a l t h o u g h , i n one e x p e r i m e n t c o m p a r i n g i n d i v i d u a l s from ponds P2 and NR, f i v e p h y s i o l o g i c a l c h a r a c t e r s were measured: m o r t a l i t y , g r o w t h r a t e s , number o f b r o o d s / f e m a l e , t o t a l e g g s / f e m a l e , t o t a l j u v e n i l e s / f e m a l e , and e g g s / b r o o d . G e n o t y p i c v a r i a t i o n d e s c r i b e d by 12 s t r u c t u r a l p r o t e i n s was measured by h o r i z o n t a l s t a r c h g e l e l e c t r o p h o r e s i s u s i n g t e c h n i g u e s s i m i l a r t o t h o s e d e s c r i b e d by S e l a n d e r e t a l . ( 1 9 7 1 ) . F i f t e e n t o t w e n t y i n d i v i d u a l s f r o m a s i n g l e c l o n e o r f r o m p o o l e d f i e l d s a m p l e s were h o m o g e n i z e d by hand w i t h a g l a s s t i s s u e g r i n d e r i n an amount o f b u f f e r (0.01 M t r i s , 0.00 1 H EDTA, and 5 x 10-5 H NADP w i t h pH a d j u s t e d t o 6.8 w i t h HCl) e q u i v a l e n t t o t h e volume o f t h e a n i m a l s . The s u p e r n a t a n t was a b s o r b e d i n t o 9 x 6 mm p i e c e s o f number 1 Whatman f i l t e r p a p e r and i n s e r t e d i n t o a s l i t i n a 12% g e l o f E l e c t r o s t a r c h ( l o t 302, H a d i s o n , Wise.) and b u f f e r . T h r e e b u f f e r t y p e s were used t o a s s a y f o r 22 l o c i ( L i O H : e s t e r a s e (ES-1, E S - 2 ) , and g l u t a m a t e o x a l a t e t r a n s a m i n a s e (GOT-1); P o u l i k : a l k a l i n e p h o s p h a t a s e (AKP-1, AKP-2, AKP-3, and AKP-4) , a c i d p h o s p h a t a s e (AP-1 and AP-2) , and l e u c i n e amino p e p t i d a s e {LAP-1, LAP-2, LAP-3, and L A P - 4 ) ; EDTA: m a l a t e d e h y d r o g e n a s e (MDH-1) , o c t a n o l d e h y d r o g e n a s e (ODH-1), s o r b i t o l d e h y d r o g e n a s e (SDH-1 and SDH-2) , x a n t h i n e d e h y d r o g e n a s e (XDH-1 and XDH-2), p h o s p h o g l u c o s e i s o m e r a s e ( P G I - 1 ) , a l d e h y d e o x i d a s e (AO-1) and i n d o p h e n o l o x i d a s e ( 1 0 - 1 ) ) . B u f f e r s and s t a i n s a r e 16 F i g u r e 2: M o r p h o l o g i c a l measures of phenotypic v a r i a t i o n . 17 18 farther described in the appendix. To determine i f there was any iotraclonal variability individual animals were also assayed using a Tsuyuki apparatus. There was no detectable difference in electrophoretic mobility between siblings, and individuals within a clone were subsequently pooled and run on the previously described systems. 19 RESULTS AND JPJLSGDSSI0J8 Genotypic, phenotypic, and environmental v a r i a t i o n are d i s c u s s e d i n t h r e e s e c t i o n s with r e s u l t s and i n t e r p r e t a t i o n of the r e s u l t s i n c o r p o r a t e d i n t o each s e c t i o n . E l e c t r o p h o r e s i s Three s p e c i e s of Daphnia were c o l l e c t e d from 22 ponds and assayed e l e c t r o p h o r e t i c a l l y f o r 12 enzymes. With the e x c e p t i o n of Daphnia pulex from NR and Box 22 (two ponds near Riske Creek, 35 km from Williams Lake) a l l i n d i v i d u a l s of Daphnia palex were monomorphic and i d e n t i c a l i n a l l p o p u l a t i o n s (Table 4). NR i n d i v i d u a l s were v a r i a b l e f o r 38% of a l l l o c i assayed. Box 22 animals were a l s o v a r i a b l e f o r the same l o c i , however too few animals were assayed f o r a c c u r a t e measurment of % polymorphic l o c i . A c t i v i t y at polymorphic l o c i PGI-1, AKP-2 and 3, and LAP 3 and H i s shown i n F i g u r e 3, however, because of the complex banding p a t t e r n s of these l o c i t h e r e has been no attempt made to measure gene f r e q u e n c i e s or % h e t e r o z y g o s i t y / i n d i v i d u a l a t v a r i a b l e l o c i . Daphnia pulex from t h r e e ponds i n the lower mainland (P2, P4, and P8) were sampled semi-monthly f o r f o u r months and a l l l o c i were monomorphic during t h i s p e r i o d . S i m i l a r l y Daphnia rosea were monomorphic i n a l l p o p u l a t i o n s although i n d i v i d u a l s from P l a c i d Lake d i f f e r e d s l i g h t l y from the other p o p u l a t i o n s i n the m o b i l i t y of s e v e r a l a l l e l e s at the AKP, 20 Table 4 : Per cent monomorphic l o c i and number of i n d i v i d u a l s assayed f o r each population and each species. specxes populations sampled number of i n d i v i d u a l s number of l o c i monomorphic l o c i (%) Daphnia UBC Research Forest: rosea Eunice 93 P l a c i d 240 Gwendoline 124 Katherine 56 UBC campus Nitobe Gardens 105 18 100% Daphnia l a e v i s Langley: Pl-A P2-A P-7 Riggs Newhouse 40 82 18 55 21 12 100% Daphnia Langley: pulex PI P2 P3 P4 P5 P6 P8 Mcleans UBC campus: Li b r a r y Burnaby: Deer Lake Williams Lake: Near Roundup Box 22 18 100% 132 250 50 342 110 41 170 96 26 90 153 52 18 62% 2 1 F i g u r e 3: E l e c t r o p h o r e t i c polymorphism a t the AKP, ES, and LAP l o c i . ES 23 AP, and LAP l o c i ( T a b l e s 4 and 5 ) . A t h i r d s p e c i e s , D a p h n i a l a e v i s were a l s o monomorphic a t a l l l o c i and i d e n t i c a l i n t h e f i v e p o p u l a t i o n s a s s a y e d ( T a b l e s 4 and 5 ) . The t h r e e s p e c i e s d i f f e r e d from one a n o t h e r a t s e v e r a l l o c i d e s c r i b e d i n T a b l e s 4 and 5 ) . I t i s d i f f i c u l t t o d e t e r m i n e whether s m a l l d i f f e r e n c e s i n e l e c t r o p h o r e t i c m o b i l i t y a r e due t o s p e c i e s d i f f e r e n c e s o r a r e a f u n c t i o n of t h e t e c h n i q u e . T h e s e d i f f e r e n c e s , however, a r e c o n s i s t e n t i n a l l g e l s and a r e assumed t o be b i o c h e m i c a l s p e c i e s d i f f e r e n c e s . F u r t h e r d e t a i l e d s t u d i e s u s i n g i s o e l e c t r i c f o c u s i n g , m u l t i d i m e n s i o n a l g e l s , o r amino a c i d c o m p o s i t i o n and s e g u e n c i n g a r e needed t o d e t e r m i n e t h e magnitude o f t h e s e d i f f e r e n c e s . Two s p e c i e s o f a n o t h e r c l a d o c e r a n , S i m o c e p h a l u s were a l s o a s s a y e d i n f o u r p o p u l a t i o n s . The two s p e c i e s , t e n t a t i v e l y i d e n t i f i e d as S. s e r r u l a t u s and S. v e t u l u s * c o u l d be r e c o g n i z e d by d i f f e r e n c e s i n m o b i l i t y and b a n d i n g p a t t e r n s a t s e v e r a l l o c i . T h e s e r e s u l t s a r e i n t e r e s t i n g n o t o n l y b e c a u s e S i m o c e p h a l u s a r e p a r t h e n o g e n e t i c a l l y r e p r o d u c i n g , but b e c a u s e 1) t h e r e a r e c o n s i s t e n t b i o c h e m i c a l d i f f e r e n c e s between s p e c i e s , and 2) an o c c a s s i o n a l h y b r i d o f t h e b i o c h e m i c a l t y p e s s u g g e s t s an i n t e r m e d i a t e o r h y b r i d of t h e two s p e c i e s ( K r e p p , u n p u b l i s h e d da t a ) . No o b v i o u s e x p l a n a t i o n e x i s t s f o r e l e c t r o p h o r e t i c h o m o g e n e i t y o f e a c h s p e c i e s i n 20 ponds i n t h e l o w e r m a i n l a n d . E l e c t r o p h o r e t i c d i f f e r e n c e s were d e t e c t e d among s p e c i e s and p o l y m o r p h i s m s o b s e r v e d i n NH and Box 22 s o i t i s u n l i k e l y t h a t t h e o b s e r v e d monomorphism o f l o w e r m a i n l a n d p o p u l a t i o n s i s a Table -> : Numerical designations for a l l e l e s measured from the origin (mm) and a l l e l e frequencies (%) for three species of Daphnia. locus JK_ rosea D. rosea D. laevis D. pulex D. pulex ( a l l pops.)* (Placid) ( a l l pops.) ( a l l pops.)**(Near Roundup) PGI-1 30 (100) 30 (100) 26 (100) 30 (100) 30 (80) 31 (20) GOT-1 43 (100) 43 (100) 40 (100) 43 (100) 43 (100) XDH-1 32 (100) 32 (100) 34 (100) 32 (100) 32 (100) 2^  30 (100) 30 (100) 28 (100) 30 (100) 30 (100) IDH-1 — — — — — — 15 (100) 15 (100) SDH-1 32 (100) 32 (100) 33 (100) 32 (100) 32 (100) 2 23 (100) 23 (100) — — 25 (100) 25 (100) AO-1 34 (100) 34 (100) 35 (100) 34 (100) 34 (100) 0DH-1 36 (100) 36 (100) 36 (100) 36 (100) 36 (100) MDH-1 30 (100) 30 (100) — — 30 (100) 30 (100) AKP-1 71 (100) 73 (100) 2 67 (100) 67 (100) 67 (100) 68 (100) polymorphic 3 57 (100) 58 (100) 57 (100) 50 (100) polymorphic 4 53 (100) 54 (100) — — ' — — AP-1 71 (100) 74 (100) 68 (100) 68 (100) 2 — 67 (100) — — 63 (100) — — ES-1 80 (100) 80 (100) 80 (100) 80 (100) polymorphic 2 76 (100) 76 (100) 76 (100) 74 (100) polymorphic LAP-1 70 (100) 70 (100) 70 (100) 70 (100) 70 (100) 2 — — — — — — — — 62 (100) 3 — — — — — — — — . 57 (36) 54 (64) 4 53 (100) 55 (100) 55 (100) 55 (100) 37 (79) 50 (21) * excluding Placid ** excluding Near Roundup 25 f u n c t i o n of t h e e l e c t r o p h o r e t i c t e c h n i q u e . A l t e r n a t i v e e x p l a n t a t i o n s f o r t h e maintenance o f v a r i a t i o n i n NR o r l a c k o f v a r i a t i o n i n a l l o t h e r ponds may r e l a t e v a r i a b i l i t y t o the t e m p o r a l and s p a t i a l s t a b i l i t y o f t h e e n v i r o n m e n t , t o d i r e c t i o n a l o r s t a b i l i z i n g s e l e c t i o n , and/or t o p o p u l a t i o n parameters such as the f r e q u e n c y of s e x u a l r e p r o d u c t i o n , r a t e o f r e p r o d u c t i o n , r a t e o f r e c r u i t m e n t from o t h e r p o p u l a t i o n s , and r a t e of m u t a t i o n . These e x p l a n a t i o n s of v a r i a b i l i t y a r e f u r t h e r c o n s i d e r e d i n t h e f i n a l d i s c u s s i o n . The e l e c t r o p h o r e t i c d i f f e r e n c e s between th e l o w e r mainland p o p u l a t i o n s and NR may s u g g e s t i n h e r e n t d i f f e r e n c e s i n the amount o f p h e n o t y p i c v a r i a t i o n i n t h e s e p o p u l a t i o n s and t h e i r a b i l i t y t o adapt t o t h e environment. For t h i s r e a s o n p h e n o t y p i c v a r i a t i o n among i n d i v i d u a l s has been compared f o r t h r e e e l e c t r o p h o r e t i c a l l y i d e n t i c a l p o p u l a t i o n s , P2, P4, and P5, and between an e l e c t r o p h o r e t i c a l l y monomorphic (P2) and e l e c t r o p h o r e t i c a l l y p o l y m o r p h i c (NR) p o p u l a t i o n . Comparison Of P2, P4, And P5; F i e l d Data P h e n o t y p i c v a r i a t i o n was measured i n Daphnia c o l l e c t e d from t h r e e ponds, P2, P4, and P5, i n t h e lower m a i n l a n d , by s c o r i n g body l e n g t h and egg number, b o t h e c o l o g i c a l l y i m p o r t a n t t r a i t s . Means, v a r i a n c e s , and 951 c o n f i d e n c e l i m i t s f o r t h e t h r e e p o p u l a t i o n s a r e g i v e n i n T a b l e 6. Even i n t h e s e t h r e e p h y s i c a l l y s i m i l a r ponds, a one-way a n a l y s i s o f v a r i a n c e (ANOVA) comparing body l e n g t h and egg number i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e s Table 6 : Estimates of the mean, variance and 95% confidence limits for body length and egg number for P2, P4, and P5 field populations. population N body length log body length egg number mean±confidence limits meaniconfidence limits meaniconfidence limits variance variance variance P2 140 1.99x10' 136.97 P4 120 2.57 xlO" 219.88 P5 109 2.30 xlO' 134.59 3.29 ± .012 6.18 xlO 3 3.40 ± .014 6.30 xl0~3 3.36 ± .012 4.45 xl0"3 8.1 ± 1.43 74.54 13.0 ± 2.24 157.26 15.3.+ 2.14 129.93 N3 ON 27 among p o p u l a t i o n s f o r both c h a r a c t e r s . This suggests e i t h e r t h a t g e n e t i c a l l y s i m i l a r organisms a r e p h e n o t y p i c a l l y f l e x i b l e , or that e l e c t r o p h o r e s i s does not measure the g e n e t i c b a s i s of phenotypic v a r i a b i l i t y . A t h i r d e x p l a n a t i o n i s t h a t the l a r g e amount of phenotypic v a r i a t i o n w i t h i n ponds may be non-genetic and i n f l u e n c e d by environmental h e t e r o g e n e i t y among p o p u l a t i o n s or by age d i f f e r e n c e s among i n d i v i d u a l s i n P2, P4, and P5. Histograms d e s c r i b i n g the d i s t r i b u t i o n of body l e n g t h ( F i g . H to 6) i n f i e l d animals i n d i c a t e extreme v a r i a t i o n among i n d i v i d u a l s i n these three p o p u l a t i o n s . I f v a r i a t i o n i n the v a r i a n c e s and means of l e n g t h and egg number i s due to environmental d i f f e r e n c e s or age v a r i a t i o n among i n d i v i d u a l s and i f the e l e c t r o p h o r e t i c v a r i a t i o n i s a good i n d i c a t i o n of o v e r a l l g e n e t i c v a r i a b i l i t y , then one would expect 1) a decrease i n v a r i a n c e i n l a b p o p u l a t i o n s measured at a s i n g l e p h y s i o l o g i c a l age, and 2) a convergence t o a common mean f o r body length and f o r egg number i n l a b reared p o p u l a t i o n s from P2, P4, and P5. 28 F i g u r e 4: D i s t r i b u t i o n o f body l e n g t h s i n f i e l d and l a b p o p u l a t i o n s o f D a p h n i a p u l e x from P2, 30 20|-0 40 Field Lab 30 20 0 1.2 1.5 2.0 Body Length rnm ,lll.limiLI»ulL . I I>Ll l l l l l J l .L — J , ! , , , , , ! , , , , ^ . 2.5 3.0 30 F i g u r e 5 : D i s t r i b u t i o n of body l e n g t h s i n f i e l d and l a b p o p u l a t i o n s of Daphnia pulex from PU. 32 F i g u r e 6: D i s t r i b u t i o n o f body l e n g t h s i n f i e l d and l a b p o p u l a t i o n s o f Daphnia p u l e x from P5. 20- Field 10 0 r Lab 30 201-10 ol 1.0 1.5 2.0 Body Length mm 2.5 34 Comparison Of P2, £4, And P5z Lab And F i e l d Data To determine the impact of age d i f f e r e n c e s among i n d i v i d u a l s and environmental h e t e r o g e n e i t y on the amount of phenotypic v a r i a b i l i t y , animals were c o l l e c t e d from f i e l d p o p u l a t i o n s P2, P4, and P5 and reared i n separate v i a l s under c o n t r o l l e d l a b c o n d i t i o n s . I n d i v i d u a l s from the f i r s t g e n e r a t i o n produced i n the l a b from each of these f i e l d animals were f u r t h e r separated i n t o i n d i v i d u a l v i a l s and s i x c h a r a c t e r s measured at the primaparous i n s t a r : body l e n g t h , body width, eye diameter, head diameter, l e n g t h o f t a i l s p i n e , and number of eggs. Histograms d e s c r i b i n g the d i s t r i b u t i o n of l e n g t h s {Fig. 4 t o 6} i n l a b animals are compared to the d i s t r i b u t i o n of l e n g t h s i n f i e l d animals. I t i s e v i d e n t even p r i o r t o s t a t i s t i c a l a n a l y s i s t h a t animals r e a r e d under c o n t r o l l e d l a b c o n d i t i o n s show c o n s i d e r a b l y l e s s v a r i a t i o n i n t h i s parameter. Variances of t h e f i e l d and l a b p o p u l a t i o n s were compared with an F t e s t and d i f f e r e d s i g n i f i c a n t l y f o r body l e n g t h and f o r egg number (Table 7) . As expected f i e l d p o p u l a t i o n s show s i g n i f i c a n t l y g r e a t e r v a r i a t i o n than l a b p o p u l a t i o n s reared from i n d i v i d u a l s from the same ponds. The r a t i o of l a b v a r i a n c e t o f i e l d v a r i a n c e (Table 8) i n d i c a t e s that of the t o t a l v a r i a t i o n observed i n the f i e l d the lab p o p u l a t i o n s c o n t a i n between 5 and 13% f o r body length and between 1 and 6% f o r egg number. However, t o a v o i d b i a s i n g the v a r i a n c e because of d i f f e r e n c e s i n mean body l e n g t h , the l o g of body l e n g t h (Lewontin, 1966) was compared. A comparison Table 7 : Comparison of lab and f i e l d variances of body length and egg number i n P2, P4, and P5. A l l values are s i g n i f i c a n t at P<.01. population body length l og body length egg number V f i e l d / V l a b V f i e l d / V l a b V f i e l d / V l a b P2 F 139 155 10.13 F 139 155 = 5 ' 6 2 F 139 155 ~ 15.89 P4 F 119 75 = 17.43 F ^ = 7.00 F 119 75 68.20 P5 F 108 163 7.91 F 1 0 8 = 3 97 163 J , y / F 108 163 ~ 31.39 LO 36 Table 8 : Comparison of lab and f i e l d variances of body length and number (% variation) in P2, P4, and P5. population body length log body length egg number V / V V /V V /V lab' f i e l d lab' f i e l d lab' f i e l d P2 P4 P5 9.9% 5.7% 12.6% 17.8% 14.3% 25.2% 6-3% 1.5% 3.2% 37 o f v a r i a n c e s o f l o g v a l u e s a l s o i n d i c a t e s i g n i f i c a n t l y l e s s r e l a t i v e v a r i a t i o n (14 - 25%) i n l a b p o p u l a t i o n s t h a n f i e l d p o p u l a t i o n s ( T a b l e 8) . , The s e d a t a s u g g e s t t h a t a l a r g e amount o f o b s e r v e d v a r i a t i o n i n a n a t u r a l p o p u l a t i o n i s a t t r i b u t a b l e t o n o n - g e n e t i c f a c t o r s : age d i f f e r e n c e s among i n d i v i d u a l s and e n v i r o n m e n t a l h e t e r o g e n e i t y . T h i s i s s i m i l a r l y t r u e i n c o m p a r i n g mean v a l u e s o f l e n g t h and egg number i n l a b and f i e l d p o p u l a t i o n s ( F i g u r e s 7 and 8 ) . T h e r e were s i g n i f i c a n t d i f f e r e n c e s i n mean body l e n g t h and mean eg g number i n t h e l a b and f i e l d p o p u l a t i o n s f r o m e a c h pond. Heans, v a r i a n c e s and 95% c o n f i d e n c e i n t e r v a l s a r e g i v e n i n T a b l e 9 f o r t h e l a b p o p u l a t i o n s . Lab r e a r e d i n d i v i d u a l s were on t h e a v e r a g e s m a l l e r w i t h fewer eggs t h a n f i e l d i n d i v i d u a l s . T h i s a g a i n may be due t o age d i f f e r e n c e s among i n d i v i d u a l s p a r t i c u l a r l y a s o l d e r i n d i v i d u a l s t e n d t o have l a r g e r c l u t c h e s t h a n p r i m a p a r o u s a d u l t s . The r e d u c t i o n i n body l e n g t h and egg number may a l s o be due t o e n v i r o n m e n t a l d i f f e r e n c e s p a r t i c u l a r l y i f t h e l a b e n v i r o n m e n t i s p o o r e r t h a n t h e f i e l d e n v i r o n m e n t . 3 8 F i g u r e 7: Means and 95% c o n f i d e n c e l i m i t s f o r body l e n g t h i n f i e l d (• ) and l a b ) p o p u l a t i o n s from P2, P I , and P5. Body Length mm CO 40 Figu r e 8: Means and 95% c o n f i d e n c e l i m i t s f o r egg number i n f i e l d <• ) and l a b (^) p o p u l a t i o n s from P2, P4, and P5. Egg Number - J . ro oi o • cn o I • 1 42 Table 9 : Estimates of the mean, variance, and 95% confidence intervals for P2, P4, and P5 lab populations. Sample sizes are indicated in parentheses. P2 (155) P4 (76) P5 (164) body length 1.56 xlO 3 ± 18 1.62 xlO 3 ± 25 1.71 xlO i 3 ±20 13.51 12.61 17.01 log length 3.19 ± .006 3.21 ± .007 3.23 + 006 1.10 xl0-3 .90 xl0-3 1.12 xlO i-3 egg number 2.67 ± .340 2.47 ± .342 2.75 + 312 4.69 2.31 4.14 log egg .106 ± .109 .223 ± .119 .185 + 101 number .479 .279 .425 variable 1 -.045 ± .011 -.004 ± .016 .046 + 013 .005 .005 . .007 •variable 2 -.021 ± .005 -.027 ± .008 .008 + 009 .001 .001 .004 variable 3 .025 ± .006 .002 -.007 ± -.008 .001 -.021 ± .007 .002 a 3 C o m p a r i s o n Of P2, PJ£, ft ng P5: L a b D a t a S i n c e age d i f f e r e n c e s and e n v i r o n m e n t a l e f f e c t s a r e r e s p o n s i b l e f o r 70 t o 90% o f t h e o b s e r v e d p h e n o t y p i c v a r i a t i o n i n f i e l d a n i m a l s , one might e x p e c t a c o n v e r g e n c e o f mean l e n g t h s and egg number among t h e t h r e e p o p u l a t i o n s i f P2, P4, and P5 a r e g e n e t i c a l l y s i m i l a r a s s u g g e s t e d by t h e e l e c t r o p h o r e s i s . T h i s i s t r u e f o r mean egg number i n which t h e r e were no s i g n i f i c a n t d i f f e r e n c e s among p o p u l a t i o n s . Egg number c o n v e r g e s t o a common mean o f 2.7 e g g s / f e m a l e ( T a b l e 9) . Mean body l e n g t h s among p o p u l a t i o n s , P2, P<4, a n d P5, however, d i f f e r s i g n i f i c a n t l y . T h i s l a c k o f c o n v e r g e n c e o f body l e n g t h may be e x p l a i n e d by a r e s i d u a l m a t e r n a l e f f e c t on body l e n g t h t o e n v i r o n m e n t a l c hange o r t o g e n e t i c d i f f e r e n c e s among p o p u l a t i o n s u n d e t e c t e d by e l e c t r o p h o r e s i s . T h e d i f f e r e n c e s i n egg number and body l e n g t h s u g g e s t i n t r i n s i c d i f f e r e n c e s i n e a c h c h a r a c t e r s * a b i l i t y t o r e s p o n d t o c h a n g e s i n t h e e n v i r o n m e n t . I t seems p o s s i b l e t h a t body l e n g t h may be i n s e n s i t i v e t o i m m e d i a t e e n v i r o n m e n t a l c h a n g e whereas e gg number may be v e r y s e n s i t i v e t o t h e i m m e d i a t e e n v i r o n m e n t and c l o s e l y a s s o c i a t e d w i t h t h e p h y s i o l o g y o f t h e p a r e n t . I f t h i s i s t h e c a s e one might e x p e c t a c o n v e r g e n c e o f mean body l e n g t h s o n l y a f t e r a number o f b r o o d s . T h i s h a s n o t been d e m o n s t r a t e d i n P2 o r i n NH i n f o u r g e n e r a t i o n s i n t h e l a b . I n c o m p a r i s o n s o f mean l e n g t h i n e a c h p o p u l a t i o n i n two s e p a r a t e e x p e r i m e n t s n e i t h e r P2 n o r NR showed any change i n mean l e n g t h 44 between experiments; l i k e w i s e there was no convergence t o a common mean. I t may be argued t h a t t h i s was not long enough f o r the p o p u l a t i o n s t o respond to the change i n the environment or t h a t t h e r e are such l a r g e d i f f e r e n c e s i n the two p o p u l a t i o n s t h a t i t i s unreasonable t o expect any convergence. More l i k e l y , however, the wide range of phenotypes r e f l e c t s a l a c k o f r i g o r o u s s e l e c t i o n i n t h e l a b environment. There i s nothing which suggests t h a t a s i n g l e genotype codes f o r a s i n g l e phenotype or a constant f i t n e s s (Kojima, 1971) i n any given environment, p a r t i c u l a r l y i f the ex p r e s s i o n of the genotype i s f a i r l y p l a s t i c . D i f f e r e n c e s i n mean length among p o p u l a t i o n s may a l t e r n a t i v e l y be due to g e n e t i c d i f f e r e n c e s undetected by e l e c t r o p h o r e s i s . T h i s p o s s i b i l i t y w i l l be f u r t h e r c o n s i d e r e d i n the next s e c t i o n based on comparisons of i n t e r - and i n t r a c l o n a l v a r i a t i o n . Comparisons Of £2, £4, And £ 5 ^ I n t e r And I n t r a c l o n a l V a r i a t i o n S p e c u l a t i o n on the source and maintenance of phenotypic v a r i a t i o n i n these organisms has r e l i e d on e x p l a n a t i o n s of environmental and e l e c t r o p h o r e t i c v a r i a t i o n i n comparison of l a b and f i e l d p o p u l a t i o n s . Since n e i t h e r o f these e x p l a n a t i o n s i s s u f f i c i e n t to account f o r a l l phenotypic v a r i a b i l i t y among pop u l a t i o n s i t i s necessary t o look a t phenotypic v a r i a t i o n a t a f i n e r l e v e l of r e s o l u t i o n , w i t h i n c l o n e s , where the genotypes of s i b l i n g s i s known. 45 I n t e r - and i n t r a c l o n a l v a r i a t i o n i s measured i n P2, P4, and P5 and t h e a n a l y s i s i s d e s c r i b e d i n f i v e s e c t i o n s : 1) p r i n c i p l e components a n a l y s i s (PCA), 2) c o m p a r i s o n o f means f o r c l o n e s w i t h i n e a c h p o p u l a t i o n , 3) c o m p a r i s o n o f means among p o p u l a t i o n s , 4) c a l c u l a t i o n o f components o f v a r i a t i o n f r o m a one way ANOVA t o d e t e r m i n e % v a r i a t i o n and a b s o l u t e v a r i a t i o n w i t h i n and among c l o n e s i n e a c h p o p u l a t i o n , and 5) c a l c u l a t i o n o f t h e components o f v a r i a t i o n f r o m a n e s t e d ANOVA t o d e t e r m i n e % v a r i a t i o n and a b s o l u t e v a r i a t i o n w i t h i n and among c l o n e s and among p o p u l a t i o n s . Ten t o t w e n t y s i b l i n g s from e a c h o f 18 t o 21 c l o n e s f r o m P2, P4, and P5 were r e a r e d i n t h e l a b i n i n d i v i d u a l v i a l s and measured a t t h e f i r s t r e p r o d u c t i v e i n s t a r f o r t h e s i x c h a r a c t e r s p r e v i o u s l y d e s c r i b e d . The m o r p h o l o g i c a l d a t a was p o o l e d by PCA and body l e n g t h , egg number, and t h e t h r e e PCA v a r i a b l e s were used t o compare t h e t h r e e p o p u l a t i o n s . P r i n c i p l e components a n a l y s i s p o o l e d t h e m o r p h o l o g i c a l measures i n t o t h r e e v a r i a b l e s a c c o u n t i n g f o r 95% o f t h e t o t a l v a r i a t i o n . V a r i a b l e 1 i s a measure o f body l e n g t h , body w i d t h , and head d i a m e t e r , and i s t h e r e f o r e an i n d i c a t o r o f body s i z e and s h a p e . V a r i a b l e s 2 and 3 a r e composed o f v a r i a t i o n due t o l e n g t h o f t a i l s p i n e and e y e d i a m e t e r w h i c h a r e p r e s u m a b l y e c o l o g i c a l l y i m p o r t a n t w i t h r e s p e c t t o p r e d a t o r a v o i d a n c e ( B r o o k s and Dodson, 1965; Z a r e t , 1972; Dodson, 1974). The PCA v a r i a b l e s and body l e n g t h were u s e d a s measures o f m o r p h o l o g i c a l v a r i a t i o n r a t h e r t h a n t h e i n d i v i d u a l m o r p h o l o g i c a l c h a r a c t e r s i n 46 a l l f u r t h e r a n a l y s e s . Each c h a r a c t e r was compared among c l o n e s w i t h i n e a c h p o p u l a t i o n u s i n g a one-way ANOVA and t h e r e were s i g n i f i c a n t d i f f e r e n c e s among c l o n e s w i t h i n e a c h p o p u l a t i o n f o r a l l c h a r a c t e r s ( F i g u r e 9 ) . T h i s r e s u l t i s c o m p l e t e l y u n p r e d i c t e d by t h e e l e c t r o p h o r e t i c d a t a . T h a t t h e r e a r e r e a l p h e n o t y p i c d i f f e r e n c e s among e l e c t r o p h o r e t i c a l l y i d e n t i c a l c l o n e s s u g g e s t s 1) t h a t t h e r e may be g e n e t i c d i f f e r e n c e s among c l o n e s o r 2) t h a t m a t e r n a l e f f e c t s among c l o n e s a r e s u f f i c i e n t t o p r o d u c e s i g n i f i c a n t d i f f e r e n c e s among c l o n e s . E a c h c h a r a c t e r was a l s o compared among t h e t h r e e p o p u l a t i o n s and t h e r e were s i g n i f i c a n t d i f f e r e n c e s i n body l e n g t h and t h e t h r e e PCA v a r i a b l e s among p o p u l a t i o n s . /There was no s i g n i f i c a n t d i f f e r e n c e i n mean egg number among P2, P4, and P5. As s u g g e s t e d i n t h e p r e v i o u s s e c t i o n t h e s e d i f f e r e n c e s may be due t o t h e r a t e o f r e s p o n s e t o c h a n g e s i n t h e e n v i r o n m e n t o r t o g e n e t i c d i f f e r e n c e s . I n f o r m a t i o n on t h e c o m p a r i s o n o f e l e c t r o p h o r e t i c a l l y i d e n t i c a l c l o n e s seems t o s u p p o r t t h e i d e a t h a t t h e r e may be g e n e t i c d i f f e r e n c e s u n d e t e c t e d by e l e c t r o p h o r e s i s w h i c h a r e r e s p o n s i b l e f o r t h e d i f f e r e n c e s i n ph e n o t y p e . Components o f v a r i a t i o n ( B e c k e r , 1967; S o k a l and R o h l f , 1969) f r o m a Model I I ANOVA were computed f o r e a c h c h a r a c t e r and p r o v i d e e s t i m a t e s o f t h e % v a r i a t i o n a t t r i b u t a b l e t o v a r i a t i o n w i t h i n and among c l o n e s . L e s s v a r i a t i o n i s e x p e c t e d among i n i n d i v i d u a l s w i t h i n c l o n e s (which a r e g e n e t i c a l l y i d e n t i c a l ) t h a n i n i n d i v i d u a l s among c l o n e s , w h i c h a l t h o u g h t h e y a r e 47 F i g u r e 9: Means and 95% c o n f i d e n c e l i m i t s f o r the three p r i n c i p a l component v a r i a b l e s of P2, P4, and P5. Variable 1 b o b b o b b b A INO  rv> A cn TJ ro 2 TJ Ol IS TJ TJ cn "D ro TJ cn Variable2 Variable 3 h H I — e - H 49 e l e c t r o p h o r e t i c a l l y i d e n t i c a l , are not n e c e s s a r i l y g e n e t i c a l l y i d e n t i c a l . T h i s e x p e c t a t i o n i s not supported, however, i n comparisons of v a r i a t i o n w i t h i n and among c l o n e s . H i t h the e x c e p t i o n of body length and body s i z e ( VI) i n P4 the gr e a t e r % v a r i a t i o n f o r each c h a r a c t e r occurs w i t h i n c l o n e s r a t h e r than among c l o n e s (Table 1 0 ) . . T h i s l a r g e v a r i a t i o n w i t h i n c l o n e s may be a f u n c t i o n of the s t a t i s t i c a l t echnique i n t h a t the w i t h i n c l o n e v a r i a t i o n i s the r e s i d u a l term of the ANOVA and i n c l u d e s any unexplained v a r i a t i o n (experimental e r r o r ) as w e l l as any 'true* w i t h i n c l o n e v a r i a t i o n . The w i t h i n c l o n e v a r i a t i o n however, may a c t u a l l y represent a l a r g e degree of v a r i a t i o n among g e n e t i c a l l y i d e n t i c a l i n d i v i d u a l s due to non-genetic f a c t o r s such as maternal e f f e c t s or m i c r o h a b i t a t d i f f e r e n c e s among v i a l s a f f e c t i n g development. A l a r g e amount of phenotypic v a r i a t i o n even among g e n e t i c a l l y i d e n t i c a l s i b s independent of s t a t i s t i c a l b i a s e s , may reduce the importance of the mean d i f f e r e n c e s among e l e c t r o p h o r e t i c a l l y i d e n t i c a l c l o n e s d e s c r i b e d i n the pr e v i o u s s e c t i o n . Based on the s i m i l a r i t y w i t h i n and among c l o n e s one would expect s i m i l a r amounts o f i n t e r - and i n t r a c l o n a l phenotypic v a r i a t i o n . Comparisons of the phenotypic v a r i a t i o n w i t h i n and among c l o n e s by an F t e s t suggest t h e r e i s no s i g n i f i c a n t d i f f e r e n c e i n i n t e r - and i n t r a c l o n a l v a r i a t i o n f o r s e v e r a l c h a r a c t e r s , s u p p o r t i n g the e l e c t r o p h o r e t i c data which i n f e r s t h a t i n d i v i d u a l s w i t h i n and among c l o n e s are e l e c t r o p h o r e t i c a l l y i d e n t i c a l . 50 Table 10 : Comparison of within and among clone variation i n P2, P4, and P5. Variances and % var i a t i o n are given. P2 P4 P5 body length* within 8.59 (62.4%) 5.40 (41.6%) 11.75 among 5.17 (37.6%) 7.58 (58.4%) 5.54 (68.0%) (32.0%) egg number within among 3.06 (64.2%) 1.68 (72.0%) 2.92 (69.5%) 1.71 (35.8%) .65 (28.0%) 1.28 (30.5%) variable 1 within among 3.60x10 ^ (67.0%) 1.77x10 (33.0%) 2.03xl0_^ (39.8%) 3.07x10 (60.2%) 4.80xl0_^ (69.9%) 2.07x10 J (30.1%) variable 2 within among .814x10";? (69.3%) ,36 xlO J (30.7%) ,954xl0_^ (80.6%) .23 xlO (19.4%) 3.18xl0_^ (85.3%) .55 xlO (14.7%) variable 3 within among L.50x10"^ (86.7%) ,23 x l 0 " J (13.3%) ,902xl0_^ (72.8%) ,35 xlO (26.2%) 1.56xl0_;r (71.9%) .61 xlO (28.1%) * Body length i s measured i n microns and variances associated with body length within and among clones are x 10 . 51 To evaluate the amount of additional variation found among populations, components of variation were also determined from a nested ANOVA p a r t i t i o n i n g variances for each character within and among clones and among populations. For a l l characters the greatest % var i a t i o n again occurs within clones and with the exception of egg number, % var i a t i o n among populations exceeds the variation among clones (Table 11). The amount of variation within and among clones i s q u a l i t a t i v e l y s i m i l a r to the amount of variation from the one-way ANOVA. Again, the variation within clones may be a function of the ANOVA, i n which the residual term includes both non-genetic and unexplained variat i o n , or the variation within clones may be genetic. As suggested by the electrophoresis these populations may be very s i m i l a r , in which case one would expect comparable amounts of morphological variation among clones and among populations. Hence, even though populations d i f f e r e d i n mean values f o r a l l characters (except egg number) they appear to be very s i m i l a r i n the amount of variation within and among clones and populations. Table 11: A comparison of v a r i a t i o n w i t h i n and among clones and among populations P2, P4, and P5. Variances and % v a r i a t i o n are l i s t e d f o r each character. v a r i a t i o n w i t h i n clones v a r i a t i o n among clones v a r i a t i o n among populations body length * 9.08 (41.0%) 5.66 (25.5%) 7.42 (33.5%) egg number 2.75 (68.5%) 1.26 (31.5%) <0 (0.0%) v a r i a b l e 1 3.81x10" •3 (45.1%) 2.18x10" •3 (25.8%) 2.45x10" •3 (29.1%) v a r i a b l e 2 1.86x10" •3 (67.5%) .374x10" •3 (13.5%) .523x10" •3 U9.0%}, va r i a b l e 3 1.42x10" •3 (57.9%) .390x10" •3 (15.9%) .645x10" •3 (26.2%) * variances associated with body length w i t h i n and among clones and among populations are x 10 . 53 Comparison Of P2, P4, And P 5 i Summary P2, P4, and P5 are s m a l l , p h y s i c a l l y s i m i l a r ponds l o c a t e d w i t h i n 10 m of one another i n Langley, B.C. A l l i n d i v i d u a l s from these t h r e e ponds are e l e c t r o p h o r e t i c a l l y monomorphic at the 16 l o c i examined. R e s u l t s based on comparisons o f f i e l d , l a b and f i e l d , and l a b pop u l a t i o n s are summarized i n Table 12. The d i f f e r e n c e s i n mean values f o r a l l phenotypic c h a r a c t e r s (except egg number i n po p u l a t i o n s ) 1) among f i e l d p o p u l a t i o n s , 2) among l a b p o p u l a t i o n s , and 3) among c l o n e s w i t h i n each popul a t i o n a l l suggest t h e r e a re r e a l d i f f e r e n c e s i n h e r e n t w i t h i n and among p o p u l a t i o n s which are not c o n s i s t e n t with the e l e c t r o p h o r e t i c data. Mean d i f f e r e n c e s i n phenotype may be l a r g e l y a t t r i b u t e d to environmental d i f f e r e n c e s among f i e l d p o p u l a t i o n s , although, t h i s i s not a p r a c t i c a l e x p l a n a t i o n i n lab r e a r e d i n d i v i d u a l s . E x p l a n a t i o n s f o r these d i f f e r e n c e s have been t e n t a t i v e l y suggested as due t o 1) slow r a t e o f change of morphological c h a r a c t e r s i n response to the l a b environment, 2) a l a r g e degree of phenotypic p l a s t i c i t y , or 3) g e n e t i c d i f f e r e n c e s undetected by e l e c t r o p h o r e s i s . Comparisons of va r i a n c e s i n d i c a t e s i g n i f i c a n t l y l e s s v a r i a t i o n i n lab po p u l a t i o n s than f i e l d p o p u l a t i o n s , presumably due to environmental e f f e c t s and age d i f f e r e n c e s among f i e l d i n d i v i d u a l s . A l a r g e v a r i a n c e p e r s i s t s , however, even among g e n e t i c a l l y i d e n t i c a l s i b s w i t h i n c l o n e s i n l a b reared p o p u l a t i o n s . T h i s suggests that these i n d i v i d u a l s are capable of a wide range of phenotypic e x p r e s s i o n from a s i n g l e genotype . 54 Table 12: Summary of the r e s u l t s from comparisons of P2, P4, and P5. P2 FIELD/LAB DATA reduced variances and means f o r length and egg number i n lab population LAB DATA e l e c t r o p h o r e t i c a l l y homogeneous FIELD DATA s i g . differences i n length and egg number among pop. i FIELD/LAB DATA reduced variances and means f o r body length and egg number i n lab pop. I LAB DATA e l e c t r o p h o r e t i c a l l y homogeneous mean body length and PCA variables s i g . d i f . among populations no s i g . d i f . i n egg number among populations FIELD/LAB DATA reduced variances and means f o r length and egg # i n lab pop. LAB DATA e l e c t r o p h o r e t i c a l l y homogeneous s i g . d i f . among clones s i g . d i f . among clones f o r s i g . d i f . among f o r a l l characters greatest % v a r i a t i o n w i t h i n clones f o r a l l characters a l l characters greatest % v a r i a t i o n w i t h i n clones f o r egg number,V2 and V3 greatest % v a r i a t i o n among clones f o r length and VI clones f o r a l l characters greatest % v a r i a t i o n w i t h i n clones f o r a l l characters greatest % v a r i a t i o n w i t h i n clones except egg number (nested ANOVA) ! variance among clones less than among pop. % v a r i a t i o n w i t h i n clones greater than among pop. which i s greater than among clones 55 The p r e v i o u s comparisons o f P2, P4, and P5 are j u s t i f i e d by the r e l a t i v e s i m i l a r i t y of t h e i r e l e c t r o p h o r e t i c data and the s i m i l a r i t y of the p h y s i c a l environment among ponds. The g e n e t i c data, p a r t i c u l a r l y the e l e c t r o p h o r e s i s and the comparisons of v a r i a n c e s , and the apparent phenotypic v a r i a t i o n suggest these p o p u l a t i o n s are i n d i v i d u a l l y b u f f e r e d and respond to changes i n the environment not by u t i l i z i n g g e n e t i c h e t e r o g e n e i t y but r a t h e r by e x h i b i t i n g phenotypic p l a s t i c i t y . T h i s c o n c l u s i o n w i l l be d i s c u s s e d r e l a t i v e to the NR data and r e l a t i v e to the s t a b i l i t y of the P2, PH, and P5 environments i n the f i n a l d i s c u s s i o n . Comparison Of P2 And NR Comparisons of P2 and NR are s i m i l a r l y j u s t i f i e d by e l e c t r o p h o r e t i c d i f f e r e n c e s between p o p u l a t i o n s and p h y s i c a l and geographic d i f f e r e n c e s between ponds to determine i f there i s more or l e s s phenotypic v a r i a t i o n i n an e l e c t r o p h o r e t i c a l l y v a r i a n t p o p u l a t i o n than an e l e l c t r o p h o r e t i c a l l y i n v a r i a n t p o p u l a t i o n . That i s , do e l e c t r o p h o r e t i c a l l y v a r i a n t p o p u l a t i o n s r e l y on l a r g e amounts of phenotypic v a r i a t i o n or on genotypic v a r i a t i o n t o adapt to environmental change? Twenty s i b s from each of f i v e c l o n e s from P2 and NR were reared i n separate v i a l s under c o n t r o l l e d l a b c o n d i t i o n s . Six c h a r a c t e r s were measured at the f i r s t r e p r o d u c t i v e i n s t a r : body l e n g t h , body width, head diamter, eye diamter, l e n g t h of t a i l 56 spine, and number of eggs. Horphological characters were pooled by p r i n c i p a l components analysis. Body length and egg number were also measured once a week i n these animals u n t i l 1/2 of both populations had died. These l a t t e r measurements provide estimates of s i x a d d i t i o n a l parameters: 1) growthrate, 2) t o t a l number of eggs/female, 3) t o t a l number of juveniles/female, 4) t o t a l number of broods/female, 5) eggs/brood, and 6) % mortality {{eggs-juveniles)/eggs). Since t o t a l eggs and t o t a l juveniles i s influenced by the number of broods produced by any female i t seems that these characeters, although important i n evaluating the f i t n e s s of the i n d i v i d u a l and of the clone, may overestimate variation. For t h i s reason i t seems p r a c t i c a l to consider r a t i o s of eggs/brood and % mortality as better i n d i c a t o r s of actual variation. Body length was measured each week for each animal and plotted against log time. Growthrates were then determined from the slope of the l i n e . Results w i l l be presented and discussed for comparisons within each population and for comparisons between populations. 57 Comparisons Of £2 And NB; I n t r a p o p u l a t i o n figsalts For £2 Re s u l t s and i n t e r p r e t a t i o n of the P2 data i n t h i s experiment are s i m i l a r to those d e s c r i b e d p r e v i o u s l y i n comparisons o f P2 r P4, and P5. 1) Daphnia from P2 were e l e c t r o p h o r e t i c a l l y monomorphic and i d e n t i c a l to animals i n the previous experiment. 2) In the primaparous i n s t a r data, a comparison of means among c l o n e s i n P2 i n d i c a t e d s i g n i f i c a n t d i f f e r e n c e s i n l e n g t h , egg number, and the three PCA v a r i a b l e s although the mean values i n t h i s experiment tended t o be l a r g e r than i n the p r e v i o u s experiment (Table 13) perhaps due t o food q u a l i t y . In comparing i n t e r c l o n a l v a r i a t i o n of growthrates, number of broods/female, t o t a l eggs, and t o t a l j u v e n i l e s , number of eggs/brood, and % m o r t a l i t y i n P2 a l l c h a r a c t e r s d i f f e r e d s i g n i f i c a n t l y among c l o n e s except growthrate and % m o r t a l i t y . From a n a l y s i s of the components of v a r i a t i o n the % v a r i a t i o n was s i g n i f i c a n t l y g r e a t e r f o r egg number, V2 and V3, number of broods, t o t a l eggs, t o t a l j u v e n i l e s , and % m o r t a l i t y w i t h i n c l o n e s than % v a r i a t i o n among c l o n e s (Tables 14 and 15). The i n t e r p r e t a t i o n of the r e s u l t s d e s c r i b e d p r e v i o u s l y i s a l s o a p p l i c a b l e to these data. 58 Table 13 : Estimates of means, variances, and 95% confidence i n t e r v a l s for P2 and NR. P2 NR . means variances means variances body length log length 1.63x 10 3 ± 32 3.21 ± .008 21.66x 10 3 1.52 x 10"' 2.19xl0 3 ±39 3.34 ± .008 35.33xl0 3 1.45 x 10 egg number log egg number 5.46 ± .63 .673 ± .054 8.40 .062 7.68 ± .64 .849 ± .038 9.36 .033 variable 1 -.135 ± .015 .005 .127 + .018 .007 variable 2 .021 ± .015 .005 .020 ± .015 .005 variable 3 -.003 ± .005 • 001 .003 ± .008 .001 growthrates log growthrates 990.9 ± 19.45 2.9 ± .02 31067.4 .01 1361 ± 85.52 3.1 ± .02 164672.7 .01 number broods log broods 7.64 ± .52 .86 ± .035 5.81 .027 10.74 ± .61 1.01 ± .034 8.53 .026 t o t a l eggs log eggs 57.99 ± 5.56 1.70 ± .056 661.7 .067 201 ± 16.04 2.26 ± .047 5827.4 .051 t o t a l juveniles log t o t a l juv. 53.03 ± 5.24 1.66 ± .062 586.3 .083 182.9 ± 13.39 2.22 ± .045 4394.6 .046 eggs/brood log eggs/brood 7.71 ± .649 .848 ± .044 9.01 .041 18.24 ± .795 1.25 ± .021 14.32 .009 % mortality log % mortality .098 ± .019 -1.44 ± .221 .008 1.05 .081 ± .009 -1.26 ± .136 .002 .416 Table 14: Comparisons of within and among clone variation in P2 and NR. Components of variation and % variation are given for each character and sample size indicated in parentheses. within P2 among within NR among body length 13.45x 56.5% IO3 10.35x 45.5% IO3 29.59x 80.7% 103 5.83x 103 19.3% egg number 6.31 70.8% 2.60 29.2% 9.10 96.6% 0.32 3.4% variable 1 2.94 56.6% 2.75 43.4% 6.27x10' 83.7% -3 1.22xl0~3 16.3% variable 2 4.01x10' 95.6% -3 0.77x10' 4.6 "3 3.66x10' 65.0% -3 1.97xl0~3 35.0% variable 3 0.46x10' 81.5% -3 0.10x10' 18.5% -3 1.23x10' 87.6% -3 0.17xl0~3 12.4% growthrates 144780 85.4% 24655 4.6% 31982 100.0% -1152 0.0% # broods 7.90 91.0% .78 9 .0% 5.14 85.9% .85 14.1% total # eggs 5243.3 87.9% 724.1 12.1% 528.8 68.6% 241.6 31.4% total # juveniles 3985.4 88.7% 507.3 11.3% 464.2 75.1% '153.8 24.9% eggs/brood 12.89 87.9% 1.77 12.1% 5.47 55.1% . 4.45 44,9% %mortality .0018 96.3% .0000 3.7% .0077 93.2% .0006 6.8% Table 15: Ratio of variances w i t h i n and among clones i n P2 and NR from untransformed and l o g a r i t h m i c a l l y transformed data. P2 NR F(81/81) witnxn among F(86/86) v / v w i t n m among body length l o g body length 1.3 1.2 * 4.17* 4.00 egg number * 2.43 , * 28.4 VI 1.26 * 5.25 V2 * 5.00 * 1.80 V3 * 5.00 6.00 * growth rate l o g growth rate * -27* -32 5.87 * 4.30 * # broods lo g # broods * 8.13 10.13* 8.50 * t o t a l eggs lo g t o t a l eggs * 2.18* 4.35 7.24 * 10.70* t o t a l j u v e n i l e s l o g t o t a l j u v e n i l e s * 3-01* 4.20 7.86 * 13.90* eggs/brood lo g eggs/brood 1.23* 1.61 7.28 * 11.00* % mortality l o g % mortality 12.83* 94.00 d i v i s i o n by 0 219.4 * P <.05 61 Comparisons Of P2 And NR: I n t r a p o p u l a t i o n l e s u l t s For NR Onl i k e P2, NR was e l e c t r o p h o r e t i c a l l y polymorphic f o r 22 to 28% o f a l l l o c i assayed. However, as i n P2, there were s i g n i f i c a n t d i f f e r e n c e s among c l o n e s f o r a l l phenotypic c h a r a c t e r s except egg number and % m o r t a l i t y . Although there were s i g n i f i c a n t d i f f e r e n c e s i n egg nummber among P2 c l o n e s , t h e r e were no d i f f e r e n c e s i n egg number among P2, P4, and P5, and the homogeneity of egg number i s unique t o t h i s s e t of c h a r a c t e r s . Estimates o f mean, va r i a n c e , and c o n f i d e n c e l i m i t s f o r the NR p o p u l a t i o n are gi v e n f o r each c h a r a c t e r i n Table 13. I n t e r c l o n a l d i f f e r e n c e s may be r e l a t e d t o e l e c t r o p h o r e t i c d i f f e r e n c e s among c l o n e s although t h i s was shown not to be the case i n P2, i e . c l o n e s i n P2 d i f f e r e d p h e n o t y p i c a l l y from one another even though they were e l e c t r o p h o r e t i c a l l y i d e n t i c a l . The v a r i a t i o n i n P2 was t e n t a t i v e l y i n t e r p r e t e d as g e n e t i c d i f f e r e n c e s among c l o n e s or as phenotypic p l a s t i c i t y a s s o c i a t e d with the s i n g l e genotype. NR i n d i v i d u a l s may a l s o be h i g h l y v a r i a b l e , however, i t seems more reas o n a b l e because of the e l e c t r o p h o r e t i c h e t e r o g e n e i t y t o a s s o c i a t e phenotypic v a r i a t i o n with genotypic d i v e r s i t y . These p o s s i b i l i t i e s have been f u r t h e r e x p l o r e d i n comparisons of i n t e r - and i n t r a c l o n a l v a r i a b i l i t y from components of v a r i a t i o n . As i n P2, the g r e a t e s t % v a r i a t i o n o f the t o t a l v a r i a n c e i n NR was w i t h i n c l o n e s r a t h e r than among clones (Table 14) and, based on comparisons o f ab s o l u t e (not %) i n t e r - and i n t r a c l o n a l v a r i a t i o n by an F t e s t , w i t h i n c l o n e 62 v a r i a t i o n was s i g n i f i c a n t l y g r e a t e r (P .05) t h a n among c l o n e v a r i a t i o n f o r a l l c h a r a c t e r s ( T a b l e 1 5 ) . One m ight e x p e c t s i m i l a r amounts o f p h e n o t y p i c v a r i a t i o n w i t h i n c l o n e s i n P2 and NR, however, a s s u g g e s t e d by t h e i n t e r c l o n a l e l e c t r o p h o r e t i c d i f f e r e n c e s i n NR t h e r e may be g r e a t e r v a r i a t i o n among c l o n e s i n NR t h a n i n P2. I n t e r p o p u l a t i o n a l c o m p a r i s o n s o f v a r i a n c e s w i t h i n and among c l o n e s between P2 and NR a r e d e s c r i b e d i n t h e n e x t s e c t i o n . T n t e r p o p u l a t i o n C o m p a r i s o n s Of £ 2 And NRr Means C o m p a r i s o n s o f means between P2 and NR i n d i c a t e s i g n i f i c a n t d i f f e r e n c e s between p o p u l a t i o n s f o r a l l c h a r a c t e r s . , O n l y means o f V2 and o f % m o r t a l i t y were g r e a t e r i n P2 and NR. F o r a l l o t h e r c h a r a c t e r s NR was s i g n i f i c a n t l y l a r g e r t h a n P2. S i n c e t h e r e were s i g n i f i c a n t d i f f e r e n c e s among P2, P4, and P5 ( p o p u l a t i o n s which a r e e l e c t r o p h o r e t i c a l l y and e n v i r o n m e n t a l l y s i m i l a r ) t h e s e d i f f e r e n c e s between NR and P2 a r e n o t u n i q u e o r u n e x p e c t e d . The t r e n d i n d i f f e r e n c e s i s , however, more d r a m a t i c and u n i d i r e c t i o n a l i n c o m p a r i s o n s o f NR and P2. I t seems p o s s i b l e t h a t t h e s e d i f f e r e n c e s may be g e n e t i c s i n c e t h e p o p u l a t i o n s a r e e l e c t r o p h o r e t i c a l l y d i s s i m i l a r and s i n c e t h e p h e n o t y p i c d i f f e r e n c e s were m a i n t a i n e d i n t h e p o p u l a t i o n s r e a r e d i n t h e l a b o v e r s e v e r a l b r o o d s . I t seems u n l i k e l y t h a t t h e s e d i f f e r e n c e s were e n v i r o n m e n t a l l y i n d u c e d . A l t e r n a t i v e l y t h e s e d i f f e r e n c e s may have been due t o a d i f f e r e n t i a l r a t e o f c h a n g e i n c h a r a c t e r s i n w h i c h a n i m a l s a r e i n c a p a b l e o f r e s p o n d i n g t o 6 3 e n v i r o n m e n t a l c h a n g e s i n o n l y a few g e n e r a t i o n s . I n t e r p o p u l a t i o n C o m p a r i s o n s Of P2 And HR: V a r i a n c e s C o m p a r i s o n s o f v a r i a n c e s i n c l u d e b o t h c o m p a r i s o n s o f % v a r i a t i o n and c o m p a r i s o n s o f a b s o l u t e v a r i a t i o n and i t i s i m p o r t a n t t o make t h i s d i s t i n c t i o n . . I n t r a p o p u l a t i o n a l c o m p a r i s o n s o f v a r i a n c e s w i t h i n and among c l o n e s a r e b a s e d on c o m p a r i s o n s o f % v a r i a t i o n o r on c o m p a r i s o n s o f t h e a b s o l u t e v a r i a n c e s . A l l i n t e r p o p u l a t i o n a l c o m p a r i s o n s a r e b a s e d on c o m p a r i s o n s o f t h e a b s o l u t e v a r i a n c e s o f t r a n s f o r m e d and u n t r a n s f o r m e d d a t a . Ho s t a t i s t i c a l c o m p a r i s o n o f % v a r i a t i o n has been made w i t h i n and among c l o n e s between p o p u l a t i o n s . V a r i a n c e s were compared w i t h an F t e s t ( T a b l e 16) and o u t o f t h e c o m p a r i s o n s o f 11 u n t r a n s f o r m e d c h a r a c t e r s o n l y t h e v a r i a n c e a s s o c i a t e d w i t h % m o r t a l i t y was g r e a t e r i n P2 t h a n NR. V a r i a n c e s a s s o c i a t e d w i t h u n t r a n s f o r m e d body l e n g t h , V3, g r o w t h r a t e , number o f e g g s / b r o o d , and t o t a l eggs and t o t a l j u v e n i l e s were g r e a t e r i n NR t h a n P2. The r e m a i n i n g c h a r a c t e r s showed no s i g n i f i c a n t d i f f e r e n c e s i n v a r i a n c e s between p o p u l a t i o n s . T h e s e d i f f e r e n c e s i n P2 and NR, however, do n o t mean t h a t NR i n d i v i d u a l s were more v a r i a b l e i n t h e e s s e n t i a l z o o l o g i c a l s e n s e t h a n P2 i n d i v i d u a l s : s i n c e NR i n d i v i d u a l s were s i g n i f i c a n t l y l a r g e r t h a n P2 i n d i v i d u a l s i t would be e x p e c t e d t h a t v a r i a n c e s would a l s o be g r e a t e r w i t h o u t any d i f f e r e n c e s i n f u n c t i o n a l v a r i a b i l i t y ( S i m p s o n , Roe, and L e w o n t i n , 1960). L e w o n t i n , however, a r g u e s t h a t by t a k i n g l o g t r a n s f o r m s o f t h e d a t a , t h e 64 Table 16: Comparison of variances f o r NR and P2 (F t e s t ) . Degrees of freedom: NR=86, P2=81 - body length * F(86/81) = 1 .63 log body length F(81/86) = 1 .05 egg number F(86/81) = 1 .11 log egg number + F(81/85) = 1 .86 va r i a b l e 1 F(86/81) = 1 .53 va r i a b l e 2 F(86/81) = 1 .14 v a r i a b l e 3 * F(86/89) =2 .54 growth rate * F(86/81) =5 .30 log growth rate * F(86/81) = 1 .62 # of generations F(86/81) = 1 .47 log # generations F(81/86) = 1 .02 t o t a l # of eggs * F(86/81) =8 .81 lo g t o t a l eggs F(81/86) = 1 .33 # of ju v e n i l e s * F(86/81) = 7.50 log # j u v e n i l e s + F(81/86) = 1 .82 # of eggs/genration * F(86/81) = 1 .59 log eggs/gen. + F(81/86) =4 .31 (eggs - juveniles)/eggs + F(81/86) =4 .31 log (eggs-juv)/eggs + F(81/86) =2 .51 * P< .05 NR being greater + P< .05 P2 being greater 65 variances, regardless of the mean, are put on the same scale and can be compared s t a t i s t i c a l l y . To estimate r e l a t i v e v a r i a b i l i t y independent of mean differences between populations log transforms of the P2 and NR data sere compared with an F test i n the two populations (Lewontin, 1 9 6 6 ) . In sharp contrast to comparisons of the o r i g i n a l data, P2 was r e l a t i v e l y more variable than NR for egg number at the f i r s t reproductive in s t a r , t o t a l number of juveniles/female, eggs/brood, and % mortality. In the eight transformed values of the t o t a l variances of log values of body length, t o t a l number of broods, and t o t a l eggs did not vary s i g n i f i c a n t l y between the two populations. Only in comparing r e l a t i v e growthrates was NR s i g n i f i c a n t l y more variable than P2 (Table 16). Having compared the t o t a l variances between P2 and NE these variances were partitioned i n t o components of v a r i a t i o n and int e r - and i n t r a c l o n a l variances of transformed data compared between populations with an F test. In comparisons of within clone variances growthrates and body length were more variable in NR than in P2 although the differences were not s t a t i s t i c a l l y s i g n i f i c a n t at P<.05. In a l l other characters P2 was more variable than NR within clones. Intraclonal variation i n P2 d i f f e r s s i g n i f i c a n t l y from NR i n t o t a l juveniles, eggs/brood and % mortality. There was no s i g n i f i c a n t difference i n number of broods/female or t o t a l eggs/female (Table 17). In comparisons of i n t e r c l o n a l variation there was a si g n f i c a n t l y greater variance in growthrate i n NR than P2. For a l l other characters except number of broods/female P2 was sign 66 Table 17: F tests comparing r e l a t i v e variances from transformed data w i t h i n and among clones between populations P2 and NR. Degrees of freedom f o r P2 = 81 and f o r NR = 86. withi n clones among clones l o g body length F(86/81) = 1. 32 F(81/86) = * 2.53 lo g growth rates F(86/81) = 1. 36 F(86/81) • ** = -9.0 lo g # of broods F(8l/86) = 1. 02 F(81/86) = 1.07 log t o t a l eggs F(8i/86) = 1. 21 F(8i/86) = * 2.98 log t o t a l j u v e n i l e F(81/86) = 1. 65 F(81/86) = * = 4.00 log eggs/brood F ( 8 i / 8 6 ) = 3. 13 F(81/86) = * 21.25 log % mortality F(81/86) = 2. 48 F(8l/86) = * -5.68 * P2 s i g n i f i c a n t l y more v a r i a b l e than NR at P < .05 ** NR s i g n i f i c a n t l y more v a r i a b l e than P2 at P< .05 67 i f i c a n t l y more v a r i a b l e than NR among c l o n e s (Table 17). Based on comparison o f t o t a l v a r i a n c e s and of i n t r a - and i n t e r c l o n a l v a r i a n c e s , P2 was g e n e r a l l y more v a r i a b l e than NR even though t h e r e was no e l e c t r o p h o r e t i c v a r i a t i o n i n the P2 p o p u l a t i o n . These r e s u l t s seem to c o r r o b o r a t e the e a r l i e r s u ggestion t h a t P2 i s i n d i v i d u a l l y homeostatic with a s i n g l e very f l e x i b l e genotype and a great d e a l of phenotypic p l a s t i c i t y . Conversely NR with g r e a t e r g e n e t i c v a r i a t i o n and l e s s phenotypic v a r i a t i o n than P2 may adapt t o the environment by p o p u l a t i o n a l homeostasis although no experiments have been done that would demonstrate d i f f e r e n t i a l f i t n e s s e s of the genotypes i n d i f f e r e n t environments. NR was a l s o capable of some phenotypic f l e x i b i l i t y as evidenced by t h e l a r g e % v a r i a t i o n w i t h i n c l o n e s and i t i s d i f f i c u l t t o e x p l a i n a d a p t a t i o n i n NR i n d i v i d u a l s by i n d i v i d u a l or p o p u l a t i o n a l b u f f e r i n g e x c l u s i v e l y . The untransformed v a r i a n c e s i n P2 and NR are f u r t h e r p a r t i t i o n e d w i t h i n and among clones and between p o p u l a t i o n s i n Table 18 i n order to look at o v e r a l l sources of v a r i a t i o n i r r e s p e c t i v e o f the p a r t i c u l a r p o p u l a t i o n . The components of v a r i a t i o n from a nested &NOVA i n d i c a t e that the g r e a t e s t % v a r i a t i o n was e i t h e r w i t h i n c l o n e s or between p o p u l a t i o n s i n c o n t r a s t with the comparisons of P2, PU, and P5 where the g r e a t e s t v a r i a t i o n f o r a l l c h a r a c t e r s was within c l o n e s . T h i s r e s u l t i m p l i e s g r e a t e r d i f f e r e n c e s between P2 and NR f o r body l e n g t h , V1, t o t a l eggs, t o t a l j u v e n i l e s , and number of eggs/brood than among P2, P4, and P5 and,although comparisons of % v a r i a t i o n do not i n d i c a t e s t a t i s t i c a l l y the magnitude of these Table 18: Comparison of v a r i a t i o n w i t h i n and among clones and between populations (P2 and NR) f o r primaparous i n s t a r . Variances and % v a r i a t i o n ( i n parentheses) are l i s t e d . v a r i a t i o n w i t h i n clones v a r i a t i o n among clones v a r i a t i o n between pop. body length 21.77 (11.53%) 8.65 (4.58%) 158.41 (83.89%) egg number 7.751 (68.95%) 1.4215 (12.64%) 2.07 (18.41%) v a r i a b l e 1 .0047 (11.59%) .00171 (4.27%) .0338 (84.14%) v a r i a b l e 2 .0038 (66.70%) .00139 (24.22%) .00052 (9.08%) v a r i a b l e 3 .0009 (86.05%) .00139 (13.95%) -.00002 (0.00% (neg.)) growth rate 94976 (55.07%) 12888.6 (7.47%) 64599 (37.46%) number of eggs 3161.6 (23.05%) 462.21 (3.37%) 10093 (73.58%) // of generations 6.6852 (55.64%) .7975 (6.64%) 4.5317 (37.72%) // of j u v e n i l e s 2430.6 (21.90%) 339.07 (3.05%) 8329.7 (75.05%) # eggs/generation 9.6169 (14.30%) 2.9251 (4.35%) 54.70 (81.35%) (eggs-juveniles)/eggs .0044 (92.89%) .00031 (6.46%) .00003 (0.65%) 00 69 d i f f e r e n c e s , t h e y do s u g g e s t t h a t t h e r e a r e r e a l d i f f e r e n c e s between P2 and NR and r e a l s i m i l a r i t i e s among P2, P4, and P5. C o m p a r i s o n Of £2 And NRj. Summary The e l e c t r o p h o r e t i c and e n v i r o n m e n t a l d i f f e r e n c e s between p o p u l a t i o n s f o r m t h e r a t i o n a l e f o r t h e c o m p a r i s o n o f P2 and NR. The two p o p u l a t i o n s d i f f e r e d e l e c t r o p h o r e t i c a l l y f r o m one a n o t h e r ; P2 was monomorphic and NR p o l y m o r p h i c . R e s u l t s a r e summarized i n T a b l e 19. Mean v a l u e s d i f f e r e d s i g n i f i c a n t l y among c l o n e s f o r most c h a r a c t e r s i n P2 and NR i n d e p e n d e n t of t h e p r e s e n c e o r a b s e n c e o f e l e c t r o p h o r e t i c v a r i a t i o n . T h e r e were s i g n i f i c a n t i n t e r c l o n a l d i f f e r e n c e s f o r a l l mean c h a r a c t e r s e x c e p t g r o w t h r a t e and % m o r t a l i t y i n P2 and egg number and % m o r t a l i t y i n NR. As i n c o m p a r i s o n s o f P2, P4, and P5 t h e r e were s i g n i f i c a n t d i f f e r e n c e s between p o p u l a t i o n s f o r mean v a l u e s o f a l l c h a r a c t e r s . D i f f e r e n c e s among c l o n e s i n e a c h p o p u l a t i o n were p r e s u m a b l y due t o m a t e r n a l e f f e c t s and r e l a t e d t o t h e p h y s i o l o g y o f t h e f e m a l e o r t o g e n e t i c d i f f e r e n c e s among c l o n e s . The l a t t e r e x p l a n a t i o n i s p a r t i c u l a r l y c o n v i n c i n g i n NR s i n c e t h e r e were e l e c t r o p h o r e t i c d i f f e r e n c e s among c l o n e s . Mean p o p u l a t i o n d i f f e r e n c e s may be due t o 1 ) d i f f e r e n c e s i n t h e p h y s i c a l and g e o g r a p h i c e n v i r o n m e n t , a s s o c i a t e d w i t h 2) t h e a b i l i t y o f m o r p h o l o g i c a l c h a r a c t e r s t o r e s p o n d t o c h a n g e s i n t h e e n v i r o n m e n t o v e r a s h o r t t i m e p e r i o d c o u p l e d w i t h a l a c k o f s t r o n g d i r e c t i o n a l s e l e c t i o n i n t h e l a b o r 3) p o t e n t i a l g e n e t i c Table 19: A summary of the genetic data from P2 and NR. P2 NR e l e c t r o p h o r e t i c a l l y homogeneous | monomorphic s i g . difference i n mean values of a l l characters among clones e l e c t r o p h o r e t i c a l l y polymorphic | heterogeneous s i g . d i f f e r e n c e i n mean values of a l l characters among clones s i g . differences i n mean values f o r a l l characters between P2 and NR greater % v a r i a t i o n w i t h i n greater % v a r i a t i o n w i t h i n clones than among clones clones than among clones P P2 greater or s i m i l a r variance i f o r a l l characters except growth rate greater % v a r i a t i o n w i t h i n clones i n NR than i n P2 greatest % v a r i a t i o n i s e i t h e r w i t h i n clones or between pop. 71 d i f f e r e n c e s , e l e c t r o p h o r e t i c or otherwise, among p o p u l a t i o n s . Components of v a r i a t i o n based on a 1-way ANOVA i n each p o p u l a t i o n i n d i c a t e g r e a t e r % v a r i a t i o n w i t h i n c l o n e s than among clones i n both P2 and NR. A comparison w i t h i n NR of a b s o l u t e i n t e r - and i n t r a c l o n a l v a r i a n c e s i n d i c a t e a l l c h a r a c t e r s are s i g n i f i c a n t l y more v a r i a b l e w i t h i n c l o n e s than among c l o n e s (P<.05). Within P2 th e r e was s i g n i f i c a n t l y g r e a t e r i n t r a c l o n a l v a r i a t i o n than i n t e r c l o n a l v a r i a t i o n f o r a l l c h a r a c t e r s except body l e n g t h and body s i z e (V1) and f o r egg number/brood. A comparison of the t o t a l v a r i a n c e , i n t e r c l o n a l v a r i a n c e , and i n t r a c l o n a l v a r i a n c e between p o p u l a t i o n s from transformed data i n d i c a t e s P2 was r e l a t i v e l y more v a r i a b l e f o r more c h a r a c t e r s than NR. A comparison of the % v a r i a t i o n and a b s o l u t e v a r i a t i o n w i t h i n each p o p u l a t i o n however suggests the p o p u l a t i o n s are p a r t i t i o n i n g the t o t a l v a r i a n c e d i f f e r e n t l y w i t h i n and among c l o n e s . There are s i m i l a r amounts of v a r i a t i o n w i t h i n and among clones i n P2 whereas the g r e a t e r v a r i a t i o n i n NR i s c o n s i s t e n t l y w i t h i n c l o n e s . Comparisons of components of v a r i a t i o n i n P2, P4, and P5, and between P2 and NR i n d i c a t e that a much gr e a t e r % of the t o t a l v a r i a t n c e i s accounted f o r between p o p u l a t i o n s of P2 and NR than among P2, P4, and P5. T h i s supports the h y p o t h e s i s , based on the e l e c t r o p h o r e t i c and environmental data, that P2, P4, and P5 are more s i m i l a r than P2 and NR and suggests the two popu l a t i o n s may r e l y on d i f f e r e n t adaptive s t r a t e g i e s b a l a n c i n g genotypic and phenotypic v a r i a t i o n . 72 C o m p a r i s o n Of P2 And N R i T e m p e r a t u r e E x p e r i m e n t A l t h o u g h a l l e x p e r i m e n t s p r e v i o u s l y d e s c r i b e d h e r e have been c a r r i e d o u t i n a s i n g l e e n v i r o n m e n t , a l l r e f e r e n c e s t o p l a s t i c i t y h a v e r e f e r r e d t o t h e a b i l i t y o f t h e o r g a n i s m t o s u r v i v e and r e p r o d u c e i n a r a n g e o f e n v i r o n m e n t s . P r e v i o u s e x p e r i m e n t s d e s c r i b e P2 as i n d i v i d u a l l y b u f f e r e d w i t h l i t t l e g e n e t i c v a r i a t i o n and l a r g e amounts o f p h e n o t y p i c v a r i a t i o n compared t o NH. NB on t h e o t h e r hand shows s i m i l a r o r l e s s p h e n o t y p i c v a r i a t i o n and i s e l e c t r o p h o r e t i c a l l y p o l y m o r p h i c . How do t h e s e p o p u l a t i o n s r e s p o n d t o d i f f e r e n t e n v i r o n m e n t s ? To f u r t h e r e v a l u a t e t h e p l a s t i c i t y o f P2 and NR e i g h t t o t e n s i b l i n g s f r o m e a c h o f t e n c l o n e s from P2 and NR h a t c h e d a t 15 C were r e a r e d a t 10, 15, and 20 C i n s e p a r a t e v i a l s . M o r p h o l o g i c a l c h a r a c t e r s and f e c u n d i t y a t f i r s t r e p r o d u c t i o n were measured and r e c o r d e d and t h e m o r p h o l o g i c a l c h a r a c t e r s p o o l e d by PCA { T a b l e 2 0 ) . S i n c e D a p h n i a a r e p o i k i l o t h e r m i c , any change i n t e m p e r a t u r e i n t h e e x t e r n a l e n v i r o n m e n t would have an e f f e c t on t h e r a t e o f enzyme r e a c t i o n s . I t has been r e p e a t e d l y d e m o n s t r a t e d t h a t t e m p e r a t u r e i s an i m p o r t a n t e n v i r o n m e n t a l p a r a m e t e r i n f l u e n c i n g f e e d i n g , g r o w t h , a n d egg p r o d u c t i o n r a t e s i n t h e s e o r g a n i s m s and one might e x p e c t v a r i a t i o n i n t h e a b i l i t y t o r e s p o n d t o t e m p e r a t u r e c h a n g e s r e l a t e d t o t h e f l e x i b i l i t y o f t h e o r g a n i s m . A n a l y s e s o f v a r i a n c e were u s e d t o compare d i f f e r e n c e s among c l o n e s w i t h i n e a c h p o p u l a t i o n a t each t e m p e r a t u r e t r e a t m e n t . Table 20: Means and variances for morphological and reproductive characters from P2 and NR reared at three temperatures. 10 C 15 C mean variance mean variance 20 C mean variance body length * 33 1.857 11.00 50 log length 33 3.268 .04105 50 egg number 33 7.212 5.9849 50 log egg number 33 .8233 .041047 50 variable 1 33 .00021 .00261 50 variable 2 33 .09574 .00346 50 variable 3 33 -.01522 .000635 50 1.519 3.180 3.080 .4611 -1.454 10.47 .00084 1.7077 .03456 .00139 -.00025 .00179 .01107 .00051 35 35 35 35 35 35 35 1.543 3.187 4.086 .5753 -.1456 -.01576 .00197 9.78 .00078 3.080 .0360 .00141 .00158 .00060 body length * 35 2.192 25.11 47 2.241 22.03 45 2.180 27.16 log body length 35 3.340 .000939 47 3.349 .00087 45 3.305 .00095 egg number 35 7.429 9.19302 47 6.787 4.736 45 5.511 3.028 log egg number 35 .8313 .03884 47 .8074 .02304 45 .7106 .0346 variable 1 35 .08553 .00388 47 .1115 .00278 45 .09170 .00319 variable 2 35 .00720 .00241 47 -.0171 .00186 45 -.04540 .00162 variable 3 35 -.01558 .00105 47 .01047 .00178 45 -.001487 .00294 3 * Means and variances associated with body length within and among clones and among populations are x 10 ; Co 74 U n l i k e t h e p r e v i o u s e x p e r i m e n t s , t h e r e was no s i g n i f i c a n t mean d i f f e r e n c e among c l o n e s i n P2 and NR. T h e r e was no s i g n i f i c a n t i n t e r c l o n a l v a r i a t i o n i n P2 a n i m a l s i n body l e n g t h , t h e t h r e e PCa v a r i a b l e s , and i n egg number a t 10 and 15 C. However P2 c l o n e s d i d d i f f e r f r o m one a n o t h e r i n number o f e g g s . T h e r e was no s i g n i f i c a n t d i f f e r e n c e among NR c l o n e s e x c e p t i n c l o n e s r e a r e d a t 10 C w h i c h were s i g n i f i c a n t l y d i f f e r e n t a t v a r i a b l e s 2 and 3. The c o n t r a s t o f t h e s e r e s u l t s w i t h t h o s e m e n t i o n e d p r e v i o u s l y may be a c c o u n t e d f o r by t h e s m a l l e r sample s i z e s i n t h i s e x p e r i m e n t . A n a l y s e s o f v a r i a n c e were a l s o u s e d t o compare d i f f e r e n c e s w i t h i n e a c h p o p u l a t i o n a t t h e t h r e e t e m p e r a t u r e s . P2 r e p l i c a t e s a t t h e t h r e e t e m p e r a t u r e s d i f f e r e d s i g n i f i c a n t l y f r o m one a n o t h e r f o r a l l m o r p h o l o g i c a l and r e p r o d u c t i v e c h a r a c t e r s . Egg number and v a r i a b l e s 2 and 3 were s i g n i f i c a n t l y d i f f e r e n t i n t h e NR r e p l i c a t e s . However, u n l i k e P2 t h e r e were no d i f f e r e n c e s i n mean body l e n g t h o r mean body s i z e (V1) i n NR a c r o s s t e m p e r a t u r e s ( T a b l e 20). P2 and NR showed e x t r e m e l y d i f f e r e n t r e s p o n s e s t o e n v i r o n m e n t a l d i f f e r e n c e s . , T h e s e d i f f e r e n c e s a r e c o n s i s t e n t w i t h o t h e r P2 and NR d a t a and w i l l be f u r t h e r d i s c u s s e d i n t h e f i n a l d i s c u s s i o n . D i f f e r e n c e s between P2 and NR a t e a c h t e m p e r a t u r e were a l s o d e t e r m i n e d by a n a l y s e s o f v a r i a n c e . Means and v a r i a n c e s f o r P2 and NR were a l s o compared t o one a n o t h e r a t e a c h t e m p e r a t u r e and t h e d a t a a r e summarized i n T a b l e 21 and F i g u r e s 10 and 11. NR i n d i v i d u a l s a r e s i g n i f i c a n t l y l a r g e r t h a n P2 i n d i v i d u a l s a t a l l 75 temperatures. However, a comparison of variances of the log length indicates neither population i s s i g n i f i c a n t l y more variable. Table 21: Comparison of means and variances i n P2 and NR at three temperatures. 10 C MEANS VARIANCES body length log body length egg number log egg number variable 1 variable 2 variable 3 ns ns ns * * P < .05 ns not s i g n i f i c a n t F(34/32) = 2.28 * F(34/32) = 1.49 F(34/32) =1.54 F(32/34) = 1.06 F(34/32) = 1.49 F(32/34) = 1.44 F(34/32) = 1.65 15 C MEANS VARIANCES ns * 20 C MEANS VARIANCES F(46/49) = 2.11 * F(46/49) = 1.03 ns F(46/49) = 2.77 * ns F(49/46) = 1.50 F(46/49) = 2.00 * F(46/49) =1.03 * F(46/49) = 3.46 * ns TEMP 10 15 20 of P2 32 49 34 of NR 34 46 44 F(44/34) = 2.78* F(44/34) - 1.22 F(34/44) = 1.02 F(34/44) = 1.04 F(44/34) = 2.26* F(44/34) = 1.03 F(44/34) = 4.91* 77 F i g u r e 10: Means and 95% c o n f i d e n c e l i m i t s f o r body l e n g t h i n P2 (° ) and NR (» ) D a p h n i a r e a r e d a t t h r e e t e m p e r a t u r e s . I Body Length (mm) bo ro 'o No 139 •a O N3 O I — H Co 79 F i g u r e 11: Means and 95% c o n f i d e n c e l i m i t s f o r egg number i n P2 (°) and NS <• ) Daphnia r e a r e d at t h r e e temperatures. 80 1 8 1 IMhh DISCUSSION Because of t h e i r reproductive biology, Daphnia which reproduce by a c y c l i c a l parthenogenesis, are useful organisms i n which to quantify genotypic and phenotypic variation and i n which to evaluate the influence of genetic and environmental variation on phenotype. I t i s important to int e r p r e t t h i s information r e l a t i v e to adaptation and f i t n e s s of parthenogenetic animals generally and of Daphnia s p e c i f i c a l l y . Because of the lack of recombination and independent assortment in ameiotic parthenogens, one might expect Daphnia to be le s s variable genotypically than sexually reproducing organisms. Using electrophoresis to measure enzyme va r i a t i o n , I observed a t o t a l lack of variation in three Daphnia species i n 20 ponds. This extreme homogeneity may be explained by the founder p r i n c i p l e : that each new population was started by a small number of females (one?) which were capable of rapidly colonizing the environment to the exclusion of a l l other genotypes. Only a profound founder e f f e c t , however, would explain the complete and consistent lack of variation in a l l ponds and the electrophoretic s i m i l a r i t y of animals from dif f e r e n t ponds within each species. This explanation, thus, seems unlikely. If i t i s assumed that enzymes are s e l e c t i v e l y important or cl o s e l y linked to s e l e c t i v e l y important characters, the electrophoretic variation i n Box 22 and NR as well as i n the 82 lower mainland p o p u l a t i o n s may be a s s o c i a t e d with the temporal and s p a t i a l s t a b i l i t y shown by the environment. The lower mainland ponds are g e o g r a p h i c a l l y s i m i l a r and exposed t o s i m i l a r e x t e r n a l environmental c o n d i t i o n s ,although they do d i f f e r s l i g h t l y i n s i z e , depth, v e g e t a t i o n , and temporal s t a b i l i t y (during the course of t h i s study one pond (P5) d r i e d up c o n s i d e r a b l y i n advance of t h e others) from one another. Thus, i t seems u n l i k e l y t h a t e l e c t r o p h o r e t i c homogeneity w i t h i n a s p e c i e s can be a t t r i b u t e d t o any p r e c i s e s p a t i a l homogeneity of p h y s i c a l c h a r a c t e r s w i t h i n the ponds. In NR, which i s p h y s i c a l l y and g e o g r a p h i c a l l y d i f f e r e n t from the lower mainland ponds, the e l e c t r o p h o r e t i c d i f f e r e n c e s (polymorphic vs. monomorphic) may be due t o both temporal and s p a t i a l heterogeneity.,However, without f u r t h e r data concerning the temporal v a r i a t i o n i n p h y s i c a l c h a r a c t e r i s t i c s of t h i s pond t h i s must remain s p e c u l a t i o n . Other e f f e c t s of environmental s t a b i l i t y on Daphnia w i l l be c o n s i d e r e d l a t e r i n t h i s d i s c u s s i o n . P o p u l a t i o n parameters, s p e c i f i c a l l y frequency of sexual r e p r o d u c t i o n , r a t e of recruitment from other p o p u l a t i o n s , and the r a t e of mutation may a l s o e x p l a i n d i f f e r e n c e s i n the amount of v a r i a t i o n i n NR and lower mainland ponds although again there i s l i t t l e i n f o r m a t i o n on these parameters. There was no evidence of change i n genotype due t o mutation or immigration i n P2, P4, or P8 which were p e r i o d i c a l l y sampled f o r f o u r months. There was no sexual r e p r o d u c t i o n i n these ponds d u r i n g t h i s time although i t i s u n l i k e l y t h a t recombination of gametes from g e n e t i c a l l y 83 i d e n t i c a l individuals with few heterozygotes would r e s u l t i n genetic changes in the offspring. The variation observed e l e c t r o p h o r e t i c a l l y i s genetic. However, whether t h i s genetic variation i s e c o l o g i c a l l y important i s unknown (lewontin, 1974). The s i g n i f i c a n t phenotypic differences among el e c t r o p h o r e t i c a l l y i d e n t i c a l clones suggest differences, possibly genetic, which are undetected by electrophoresis. Ideally, to evaluate the importance of electrophoretic v a r i a b i l i t y i t i s necessary to l i n k the function of the enzyme to the environment and to demonstrate selection acting at the enzyme l e v e l . Since t h i s i s impractical i n most studies, i t i s possible a l t e r n a t i v e l y to correlate i d e n t i f i a b l e enzyme types with the environment, regardless of the s p e c i f i c function of the enzyme, as i n ftyena or with data on phenotypic variation as done in t h i s study. Clearly, detailed studies of in d i v i d u a l responsiveness to different environments, population dynamics, and environmental fluctuations need to be coupled to determine the mechanisms for maintaining these di f f e r e n t genetic structures i n di f f e r e n t populations. In populations of Daphnia there are differences i n means of morphological and physiological t r a i t s among populations regardless of the electrophoretic or geographical s i m i l a r i t y of the populations. Likewise there are differences i n means of some characters among clones within populations, again independent of electrophoretic and environmental s i m i l a r i t y . There i s no obvious explanation for these mean differences i n such s i m i l a r 84 p o p u l a t i o n s or c l o n e s . These d i f f e r e n c e s suggest t h a t e l e c t r o p h o r e t i c s i m i l a r i t y does not n e c e s s a r i l y provide a good i n d i c a t i o n of phenotypic s i m i l a r i t y . Mean phenotypes may be i n f l u e n c e d by the environment or by g e n e t i c d i f f e r e n c e s undetected by e l e c t r o p h o r e s i s ; however, with no i n f o r m a t i o n on how s e l e c t i o n operates on these phenotypes, mean d i f f e r e n c e s among c l o n e s and between p o p u l a t i o n s p r o v i d e l i t t l e i n f o r m a t i o n on the adaptive s t r a t e g i e s of these organisms. The e x i s t e n c e of mean d i f f e r e n c e s among c l o n e s and among po p u l a t i o n s f o r some c h a r a c t e r s and not f o r other suggests t h a t d i f f e r e n t c h a r a c t e r s respond d i f f e r e n t l y both i n degree and i n r a t e of change. Bradshaw (1965) argues t h a t p l a s t i c i t y , the amount by which the i n d i v i d u a l genotype can be modified by i t s environment, i s s p e c i f i c f o r each c h a r a c t e r and s p e c i f i c i n r e l a t i o n to p a r t i c u l a r environmental i n f l u e n c e s . I t i s d i f f i c u l t however t o assess whether c h a r a c t e r s i n Daphnia are v a r y i n g independently of one another. M o r p h o l o g i c a l c h a r a c t e r s were r e l a t i v e l y r e l a t e d based on c o r r e l a t i o n c o e f f i c i e n t s among c h a r a c t e r s (Table 22). There was no evidence, however, t h a t the c h a r a c t e r s a s s o c i a t e d with body s i z e and those a s s o c i a t e d with predator avoidance were more or l e s s p l a s t i c or varying independently of one another.. S i m i l a r l y t h e r e was no obvious d i f f e r e n c e i n the amount o f p l a s t i c i t y i n m o r p h o l o g i c a l and p h y s i o l o g i c a l c h a r a c t e r s , although egg number showed gre a t e r conformity among p o p u l a t i o n s (P2, P4, and P5) and among clones (NR) than other c h a r a c t e r s . T h i s may imply t h a t egg number i s an extremely p l a s t i c c h a r a c t e r capable of responding i n a very sh o r t time to changes i n the environment. Egg number i s most Table 22: Comparisons of morphological characters in'P2 and NR. correlation matrix N=I69 body length 1.000 body width .986 1.000 t a i l spine .873 .876 1.000 eye diameter .343 .333 . 193 1.000 head width .976 .973 .873 .287 Comparisons of morphological characters i n P2,P4,andP5. correlation matrix N=391 body length 1.000 body width .908 1.000 t a i l spine .061 .057 1.000 eye diameter .320 .239 -.009 1.000 head width .914 .858 .084 .287 86 l i k e l y c o r r e l a t e d w i t h body l e n g t h and w i t h the g e n e r a l p h y s i o l o g y o f t h e p a r e n t . In t h i s r e s p e c t i t does seem t o d i f f e r at l e a s t q u a l i t a t i v e l y from the o t h e r c h a r a c t e r s examined i n i t s response t o e n v i r o n m e n t a l change. Comparisons of e l e c t r o p h o r e t i c v a r i a b i l i t y , v a r i a n c e s among p o p u l a t i o n s , and components o f v a r i a t i o n w i t h i n and among c l o n e s suggest d i f f e r e n c e s between P2 and NR and a n e g a t i v e c o r r e l a t i o n o f g e n e t i c and p h e n o t y p i c v a r i a b i l i t y i n p o p u l a t i o n s o f 23£]lQ.i§ E S l e x • p 2 Daphn i a w i t h l e s s e l e c t r o p h o r e t i c v a r i a b i l i t y and more r e l a t i v e p h e n o t y p i c v a r i a t i o n t h a n NR a n i m a l s p a r t i t i o n e d t h e t o t a l v a r i a n c e e q u a l l y w i t h i n and among c l o n e s and among p o p u l a t i o n s . NR Daphnia were e l e c t r o p h o r e t i c a l l y p o l y m o r p h i c and showed l e s s a b s o l u t e p h e n o t y p i c v a r i a t i o n between p o p u l a t i o n s , w i t h i n c l o n e s , and among c l o n e s than P2 w i t h t h e g r e a t e s t % v a r i a t i o n i n NR w i t h i n c l o n e s . S i m i l a r d a t a have been d e s c r i b e d i n Avena b a r b a t a and A. f a t u a ( M a r s h a l l and A l l a r d , 1970) and i n D r o s o p h i l a ( C a r s o n , 1965) and i n t e r p r e t e d r e l a t i v e t o t h e c o n t r i b u t i o n o f i n d i v i d u a l h omeostasis and g e n e t i c polymorphism t o a d a p t a b i l i t y o f the p o p u l a t i o n . L e w o n t i n (1957) p o i n t s o u t t h a t p o p u l a t i o n s may adapt t o change i n the environment e i t h e r 1) by p o p u l a t i o n a l h o m e o s t a s i s , where t h e g e n o t y p i c c o m p o s i t i o n of t h e p o p u l a t i o n may be f l e x i b l e , 2) by i n d i v i d u a l h o m e o s t a s i s where each i n d i v i d u a l i s f i t i n a number of e n v ironments by b e i n g p h e n o t y p i c a l l y p l a s t i c , o r 3) by some c o m b i n a t i o n o f i n d i v i u d a l f l e x i b i l i t y and g e n e t i c d i v e r s i t y t h a t maximizes f i t n e s s . These d i f f e r e n c e s i n P2 and NR may be s i m i l a r l y i n t e r p r e t e d as 87 d i f f e r e n t adaptive strategies though they d i f f e r i n degree rather than kind. Whether these differences are quantitative or q u a l i t a t i v e and whether they are pathological or s t r a t e g i c are undetermined and i t seems more p r a c t i c a l to look at P2 and Ne as populations which need to deal with d i f f e r e n t amounts of seasonal change and, within any i n d i v i d u a l l i f e t i m e , similar amounts of environmental change. This w i l l be done by 1) further estimating environmental s t a b i l i t y of P2 and NR from f i e l d data, 2) measuring f i t n e s s i n populations of Daphnia £ulex reared i n the lab at three di f f e r e n t temperatures, and 3) by interpreting genetic and phenotypic v a r i a b i l i t y r e l a t i v e to s e l e c t i o n and s t a b i l i t y in a model environment. Environmental S t a b i l i t y Of P2 And NR A further i n t e r p r e t a t i o n of environmental s t a b i l i t y of P2 and NR i s necessary before continuing the discussion of the genetic and phenotypic data in Dafikfiia . However, the interpretation i s primarily speculative, based only on f i e l d observations and the data described i n Tables 2 and 3. The P2, P4, and P5 environment may or may not be stable over short periods of time. The three ponds are a l l small and f a i r l y shallow and thus may be sensitive t o any external environmental change. However, the ponds are well shaded and there i s a large reserve of ground water which may s u f f i c i e n t l y buffer these ponds against any severe short term changes i n volume. Long term seasonal changes in the lower maniland are not 88 p a r t i c u l a r ! y dramatic and p o p u l a t i o n s may be a b l e to s u r v i v e by phenotypic p l a s t i c i t y alone. Populations of Daphnia i n the Peterson ponds, however, are temporary, dying out i n t h e f a l l e i t h e r due to the a c t u a l disappearance of the ponds as they dry up or by some other environmental s t i m u l u s , presumably decreased temperature or amounts of food, or an i n c r e a s e d p o p u l a t i o n d e n s i t y as a consequence of the s m a l l e r volume of the pond. Daphnia i n these ponds seem t o respond to these l o n g term changes a s s o c i a t e d with the disappearance of these ponds by forming e p h i p p i a l eggs r a t h e r than by g e n e t i c changes i n the p o p u l a t i o n or by phenotypic f l e x i b i l i t y . I t seems l o g i c a l t h a t i f t h e r e i s no p o s s i b i l i t y of continued s u r v i v a l i n a pond, then an i n d i v i d u a l i n c r e a s e s i t s f i t n e s s by producing e p h i p p i a l eggs which w i l l i n c r e a s e p r o b a b i l i t y of progeny i n the next season. There are probably very few s h o r t term changes o c c u r r i n g i n NR because of the l a r g e s i z e and depth of t h i s pond. Comparisons of temperature p r o f i l e s from two s i m i l a r l a k e s i n Riske Creek i n d i c a t e much l e s s v a r i a t i o n i n d a i l y minimum/maximum temperatures at depths g r e a t e r than 30 cm than at shallow depths (surface) (Toppings, 1969; Jansonn, 1971). I f the Daphnia pulex a r e l o c a t e d at depths g r e a t e r than 30 cm then they probably do not experience ouch environmental v a r i a b i l i t y over a s h o r t p e r i o d of time. I t i s not known whether Daphnia s u r v i v e throughout the winter i n NR though i t seems u n l i k e l y s i n c e the pond f r e e z e s over. However, s i n c e the pond i t s e l f i s permanent, changes i n the g e n e t i c s t r u c t u r e o f the p o p u l a t i o n may be adaptive i n s u r v i v i n g long term environmental changes. 89 These d i f f e r e n c e s i n genotypic and phenotypic v a r i a t i o n may suggest d i f f e r e n c e s i n the a b i l i t y of these organisms to adapt to d i f f e r e n t environments. A d a p t a b i l i t y i n these p o p u l a t i o n s has been d e s c r i b e d r e l a t i v e to t h e environmental s t a b i l i t y and to t h e i r response t o temperature. P2 and NR show extremely d i f f e r e n t responses to d i f f e r e n t temperatures, however, both responses may be e x p l a i n e d by phenotypic p l a s t i c i t y . Phenotypic f l e x i b i l i t y a s s o c i a t e d with a s i n g l e genotype i n P2 may be r e s p o n s i b l e f o r the observed d i f f e r e n c e s i n means among g e n e t i c a l l y i d e n t i c a l r e p l i c a t e s . In e l e c t r o p h o r e t i c a l l y polymorphic i n d i v i d u a l s i n NR, however, phenotypic f l e x i b i l i t y by extreme developmental c a n a l i z a t i o n may have been r e s p o n s i b l e f o r convergence of means at d i f f e r e n t temperatures. I t i s d i f f i c u l t t o determine i f the divergence i n P2 and t h e convergence i n NR a c t u a l l y c o n f e r an a d a p t i v e advantage to e i t h e r p o p u l a t i o n . In comparisons of mean adaptive values (H) determined from mean number o f s u r v i v o r s and mean number o f eggs at a l l temperatures NR was g r e a t e r than P2 f o r both c h a r a c t e r s . P2 however has the g r e a t e r v a r i a n c e i n f i t n e s s than NR (Table 23) . Greater mean f i t n e s s g e n e r a l l y a s s o c i a t e d with a low v a r i a n c e i n f i t n e s s has s i m i l a r l y been observed i n D r o s o p h i l a pseudoobscura (Lewontin, 1957) from a s i n g l e p o p u l a t i o n where homozygotes showed l e s s average f i t n e s s and gre a t e r v a r i a n c e i n f i t n e s s than heterozygotes. T h i s does not i n d i c a t e which p o p u l a t i o n o f Daphnia or which genotype of D r o s o p h i l a i s more f i t ; however, i t does i n d i c a t e two types of 9 0 Table 23: Means and variances f o r f i t n e s s based on number of survivors and number of eggs from P2 and NR i n the temperature experiment. NUMBER OF SURVIVORS 10C 15C 20C W " UJ NR 35 47 44 42.0 78.0 P2 33 51 35 39.7 194.7 TOTAL EGGS 10C 15C 20C W NR 240 301 242 261 . 2402 P2 232 149 153 178 4382 Q u a l i t a t i v e comparison of mean f i t n e s s and variance i n f i t n e s s i n P.2 and NR MEAN FITNESS VARIATION IN FITNESS NR HIGHER LOWER P2 LOWER HIGHER 91 f i t n e s s , one associated with larger means and smaller variances,the other with smaller means and larger variances., Since there i s l i t t l e environmental data available a hypothetical model r e l a t i n g phenotypic and genotypic variation to selection and to the temporal s t a b i l i t y of the environment i s described. The apparent trade-off of i n d i v i d u a l homeostasis and genetic polymorphism described previously i n P2 and NR may be closely associated with s e l e c t i v e pressures on the organism. I f the NR environment i s such that a single phenotype i s advantageous, a l l individ u a l s regardless of their electrophoretic genotype w i l l tend to converge on that optimal phenotype, either by phenotypic f l e x i b i l i t y associated with developmental canalization or by selection f o r genotypes coding for that phenotype. This convergence would account f o r the for the reduced variance among clones and i n the population. In t h i s model any i n t r a c l o n a l v a r i a t i o n observed i n t h i s study i s attributed to experimental error. If i n P2 the environment i s l e s s r e s t r i c t i v e and there i s l i t t l e s e l e ction f o r a single phenotype, then t h i s would account for greater absolute variation i n the population and the d i s t r i b u t i o n of variances within and among clones. The severity of sel e c t i o n in these populations may be influenced by the environmental s t a b i l i t y . , I f changes i n the environment are of the same or shorter duration than the generation time of the organism adaptation can only take place by individual homeostasis. The organism cannot respond to short term environmental changes by genetic changes unless they are 92 a s s o c i a t e d w i t h t h e development o f the organism. I f , however, changes i n t h e environment take l o n g e r t h a n t h e g e n e r a t i o n time a d a p t a t i o n may t a k e p l a c e by g e n e t i c changes i n t h e p o p u l a t i o n . . I f t h e measures o f g e n o t y p i c and p h e n o t y p i c v a r i a t i o n i n P2 and NR are a r e a l i n d i c a t i o n of the a d a p t i v e s t r a t e g i e s t h e n one might assume P2 i s w e l l adapted t o s h o r t term e n v i r o n m e n t a l f l u c t u a t i o n s w i t h l i t t l e a b i l i t y t o adapt t o s e v e r e l o n g term changes. NR may a l s o be s u f f i c i e n t l y i n d i v i d u a l l y b u f f e r e d t o adapt t o s h o r t term changes. F u r t h e r , because o f i t s g e n e t i c d i v e r s i t y t h e NR p o p u l a t i o n i s a l s o b u f f e r e d o v e r l o n g term e n v i r o n m e n t a l d i f f e r e n c e s . P o p u l a t i o n a l h o m e o s t a s i s i s dependent on g e n e t i c v a r i a b i l i t y i n t h e p o p u l a t i o n and i s m a i n t a i n e d i n p a r t by s e x u a l r e p r o d u c t i o n . S i n c e p a r t h e n o g e n e t i c organisms cannot n e c e s s a r i l y r e l y on r e c o m b i n a t i o n and random a s s o r t m e n t t o m a i n t a i n g e n e t i c d i v e r s i t y p h e n o t y p i c v a r i a b i l i t y and i n d i v i d u a l h omeostasis seem t o be a more r e l i a b l e means of a d a p t i n g t o the environment. T h i s has been proposed f o r p o p u l a t i o n s of t h e s n a i l , Rumina d e c o l l a t a and f o r p o p u l a t i o n s o f w i l d o a t s , Avena b a r b a t a which are e l e c t r o p h o r e t i c a l l y homogenous and p h e n o t y p i c a l l y v a r i a b l e . However, a l l o t h e r s t u d i e s measuring g e n e t i c v a r i a b i l i t y i n p a r t h e n o g e n e t i c p o p u l a t i o n s r e p o r t l a r g e amounts o f v a r i a t i o n a p p a r e n t l y u n a f f e c t e d by the l a c k o f s e x u a l r e p r o d u c t i o n and m a i n t a i n e d by s e l e c t i o n or some o t h e r mechanism. These d i f f e r e n c e s i n t h e amount of v a r i a b i l i t y i n p a r t h e n o g e n e t i c organisms seem to suggest t h a t v a r i a t i o n may n o t be as r i g o r o u s l y l i n k e d t o t h e mode o f r e p r o d u c t i o n as t o 93 s e l e c t i o n and environmental s t a b i l i t y , £!&bnia are apparently capable o f l a r g e amounts of phenotypic f l e x i b i l i t y and both g e n e t i c and phenotypic v a r i a b i l i t y seem t o be more c l o s e l y a s s o c i a t e d with environmental parameters than r e p r o d u c t i v e s t r a t e g y . Hebert (1974) does, however, presents data from temporary and permanent p o p u l a t i o n s that suggest g e n e t i c v a r i a t i o n i s c l o s e r to Hardy-Weinberg e q u i l i b r i u m i n temporary ponds, which presumably undergo more fre q u e n t s e x u a l r e p r o d u c t i o n . an a l t e r n a t i v e a d a p t a t i o n to both phenotypic f l e x i b i l i t y and g e n e t i c polymorphism i n Daphnia i s t h e i r a b i l i t y to produce o v e r w i n t e r i n g e p h i p p i a l eggs. Regardless of the q e n e t i c consequences of s e x u a l r e p r o d u c t i o n i n these i n d i v i d u a l s the formation of e p h i p p i a l eqqs provides a means o f s u r v i v i n g d i f f i c u l t times i n these ponds. 94 LITERATURE CITED B a c c i , G., G. C o g n e t t i , A.C. V a c c a r i . 1961. E n d o m e i o s i s and s e x d e t e r m i n a t i o n i n D a p h n i a p u l e x . E x p e r i e n t i a 15: 5 0 5 , ; B a n t a , A.M. 1939. 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G e n o t y p i c f r e g u e n c i e s i n permanent p o p u l a t i o n s . G e n e t i c s 77:323-334. H e b e r t , P.D.N. 1974. Enzyme v a r i a b i l i t y i n n a t u r a l p o p u l a t i o n s o f D aphnia I§.aH§. H I . G e n o t y p i c f r e g u e n c i e s i n i n t e r m i t t e n t p o p u l a t i o n s . G e n e t i c s 77:335-341. . H e b e r t , P.D.N. And B.D. Ward. 1972., I n h e r i t a n c e d u r i n g p a r t h e n o g e n e s i s i n D a p h n i a magna . G e n e t i c s 71:639-642. H u t c h i n s o n , G.E. 1967. A T r e a t i s e on L i m n o l o g y . W i l e y and Sons. New York. J a c o b s , J . 1966. P r e d a t i o n and r a t e o f e v o l u t i o n i n c y c l o r a o r p h i c D a p h n i a . V e r h . I n t . V e r . L i m n o l . 16:1645-1652. J a i n , S.K. And S.D. M a r s h a l l . 1967. P o p u l a t i o n s t u d i e s i n p r e d o m i n a n t l y s e l f p o l l i n a t i n g s p e c i e s . X. V a r i a t i o n i n n a t u r a l p o p u l u l a t i o n s o f Avena f a t u a and Avena b a r b a t a . Am. Nat. 101:19-33. J a n s s o n , A.B.I. 1971. S t r i d u l a t i o n and i t s s i g n i f i c a n c e i n t h e wate r b u g genus C e n o c o r i x a . Ph.D. T h e s i s . U n i v e r s i t y o f o f B r i t i s h C o l u m b i a . K o j i m a , K. 1971. I s t h e r e a c o n s t a n t f i t n e s s f o r a g i v e n g e n o t y p e ? No! E v o l . 25:281-285. L e v i n s . R. 1965. T h e o r y o f f i t n e s s i n a h e t e r o g e n o u s e n v i r o n m e n t . V. O p t i m a l g e n e t i c s y s t e m s . G e n e t i c s 52:891-904. L e v i n s , B. 1968. E v o l u t i o n i n a C h a n g i n g E n v i r o n m e n t . P r i n c e t o n U n i v e r s i t y P r e s s . 120pp. , L e w o n t i n . B.C. 1957. The a d a p t a t i o n s o f p o p u l a t i o n s t o v a r y i n g e n v i r o n m e n t s . C o l d s p r i n g H a r b o r Symp. Quant. B i o l . 22:395-408. 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E x p e r i m e n t e l l e und c y t o l o g i s c h e u n t e r s u c h u n g e n u b e r den g e n e r a t i o n - w e c h e l s e l d e r c l a d o c e r a n . Z o o l . J b . P h y s i o l . 56:323-388. N o r t h c o t e , T.G. And R. C l a r o t t o . 1975. L i m n e t i c m a c r o z o o p l a n k t o n and f i s h p r e d a t i o n i n some c o a s t a l B.C. , L a k e s . , V e r h . I n t e r n a t . V e r e i n . L i m n o l . 19:237 8-2393. P a r k e r , E.D. And R.K. S e l a n d e r . 1976. The o r g a n i z a t i o n o f g e n e t i c d i v e r s i t y i n t h e p a r t h e n o g e n e t i c l i z a r d , C n e m i d o p h o r u s t e s s e l a t u s . G e n e t i c s 81:791-805. P e r s i g , R.M. 1975.,Zen and t h e A r t o f M o t o r c y c l e M a i n t e n a n c e . Bantam Books. New Y o rk. S e l a n d e r , R.K. 1976. G e n i e v a r i a t i o n i n n a t u r a l p o p u l a t i o n s . I n M o l e c u l a r E v o l u t i o n . Ed. F . , A y a l a . P.21-45, S e l a n d e r , R.K., M.H. S m i t h , S.Y. Yang, W.E. J o h n s o n , and J.B. G e n t r y . B i o c h e m i c a l p o l y m o r p h i s m i n t h e genus Peromyscus. I . V a r i a t i o n i n t h e o l d - f i e l d mouse P e r o m y s c u s p o l i o n o t u s . S t u d i e s i n G e n e t i c s . V I . U n i v . T e x a s P u b l . 7103:49-90. S e l a n d e r , R.K. And D. Kaufman. 1973. G e n e t i c v a r i a b i l i t y and s t r a t e g i e s o f a d a p t a t i o n i n a n i m a l s . P r o c . Nat. Acad. S c i . 70:18 75-1877. S e l a n d e r , R.K.and D.W. Kaufman. 1973. S e l f - f e r t i l i z a t i o n and g e n e t i c p o p u l a t i o n s t r u c t u r e i n a c o l o n i z i n g l a n d s n a i l . P r o c . Nat . Acad. S c i . 70:1186-1190. Simpson, G.G., A. Roe, and R.C. L e w o n t i n . 1960. Q u a n t i t a t i v e Z o o l o g y . H a r c o u r t , B r a c e , and Co., I n c . New Y o r k . S m i t h , M.Y. And A., F r a s e r . 1976. 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APPENDIX Buffers: LiOH Stock A (electrode buffer) pH = 8.1 2.4 g LiOH 23.8 g b o r i c a c i d make up to 2 1 with d i s t i l l e d water Stock B pH = 8.4 12.4 g TRIS b u f f e r 3.2 g c i t r i c a c i d make up to 2 1 with d i s t i l l e d water Gel b u f f e r 25 ml Stock A + 215 ml Stock B 250 v o l t s f o r 3 hours Poulik Electrode b u f f e r pH 8.12 37.10 g b o r i c a c i d 4.8 g NaOH make up to 2 1 with d i s t i l l e d water Gel b u f f e r pH = 8.62 18.42 g TRIS b u f f e r 2.10 g c i t r i c a c i d monohydrate make up to 2.1 with d i s t i l l e d water 250 v o l t s f o r 3 hours EDTA Gel and Electrode buffers pH = 9.00 42.2 g TRIS b u f f e r 1.2 g EDTA 2.0 g b o r i c ac i d make up to 4 1 with d i s t i l l e d water add 20 mg NAD to gel b u f f e r when making gel 350 v o l t s f o r 4 hours Stains: Used with LiOH: ES incubate i n TRIS. malate: T r i s malate 100 ml (see LAP stain) f a s t blue RR 20 mg Na napthyl acetate 10 ml (lOOmg i n 5 ml water + 5 ml acetone) GOT TRIS (0.1 M) pH = 8.5 100 ml as p a r t i c a c i d 4.40 mg k e t o g l u t a r i c a c i d 240 mg f a s t blue BB 80 mg pyri d o x a l 5' phosphate 2 mg Used with Poulik: AKP TRIS HC1 (0.1 M) pH = 8.5 100 ml PVP 500 mg f a s t blue BB 100 mg napthyl a c i d phosphatase 100 mg MgCl2 60 mg MnCl2 60 mg NaCl 2 g AP incubate f o r 30 min. i n 0.5 M b o r i c a c i d , then to: 0.125 M sodium acetate pH=5.0 100 ml PVP 500 mg Na-napthyl a c i d phosphatase 100 mg f a s t blue BB 100 mg LAP incubate f o r 30 min. i n 0.5 M b o r i c a c i d , then to: TRIS malate 100 ml 12.1 g TRIS 11.6 g maleic a c i d 1.0 N NaOH 1 ml make up to 1 1 with d i s t i l l e d water f a s t black K 20 mg Na L-leucine 20 mg Used with EDTA: MDH ODH SDH XDH PGI AO TRIS HCl 0.1 M pH='8.5 malic acid 50 mg NAD 20 mg KC1 10 mg PMS 2 mg TRIS HCl 0.1 M pH=8.5 100 ml NAD 25 mg NBT 20 mg Octanol 5 ml PMS 2 mg TRIS HCL 0.1 M pH=8.5 100 ml NAD 25 mg NBT 20 mg D- s o r b i t o l 500 ug PMS 2 ug TRIS HCl 0.1 M pH.7-7.4 100 ml hypoxanthine 100 mg NAD 25 mg PMS 5 mg NBT 20 mg KCL 100 mg TRIS HCl 0.05 M pH 8.5 100 ml MgCl2 100 mg fructose-6-phosphate 25 mg NADP 15 mg MTT 25 mg G-6-PDH 25 units PMS 10 mg TRIS HCl 0.05 M pH 8.5 100 ml benzaldehyde 1 ml NBT 20 mg NAD 25 mg EDTA 10 mg PMS 10 mg 

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