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Genetic relationships among threespine sticklebacks, Gasterosteus aculeatus Withler, Ruth Elinor 1980

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GENETIC RELATIONSHIPS AMONG THBEESPINE STICKLEBACKS GAST EBOSTEUS &CULEA!DO§ by BOTH ELI NOB SITHLEB B. Sc. (Hons.), U n i v e r s i t y o f B r i t i s h Columbia, 1976 A THESIS SUBMITTED IN PABTIAL FULFILMENT OF THE REQUIREMENTS FOB THE DEGBEE OF MASTER OF SCIENCE i a THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s t h e s i s as conforming to the r e q u i r e d s t a n d a r d THE UNIVEBSITI OF BHITISH COLUMBIA A p r i l 1980 Q Ruth E l i n o r Withler, 1980 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ZOOLOGY The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date April 23. 1980 i i a b s t r a c t Threespine s t i c k l e b a c k s (Gasterosteus aculeatus) i n h a b i t both marine and freshwater environments along the P a c i f i c coast o f North America, In t h i s study, s t i c k l e b a c k s c o l l e c t e d from 73 l o c a t i o n s on Vancouver I s l a n d , the S e c h e l t P e n i n s u l a and the lower F r a s e r R i v e r V a l l e y of B r i t i s h Columbia, and s i x s i t e s i u northwestern Washington S t a t e , were assayed by s t a r c h g e l e l e c t r o p h o r e s i s i n order to examine r e l a t i o n s h i p s among and between marine (trachurus) and freshwater ( l e i u r u s ) populations.,,, S i x enzymes, coded f o r by e i g h t g e n e t i c l o c i , were examined. Of these, two aere monomorphic f o r the same a l l e l e i n a l l p o p u l a t i o n s , the remainder were polymorphic t o v a r y i n g degrees. ; L a b o r a t o r y b r e e d i n g s t u d i e s employing both marine and freshwater f i s h as parents confirmed the g e n e t i c i n t e r p r e t a t i o n of observed v a r i a b i l i t y i n isozyme banding p a t t e r n s . One of the monomorphic enzytaes, i s o c i t r a t e dehydrogenase, e x h i b i t e d a s e x u a l l y dimorphic isozyme p a t t e r n . L e v e l s of polymorphism and h e t e r o z y g o s i t y were s l i g h t l y higher than average, bat w i t h i n the range of those c h a r a c t e r i z i n g other v e r t e b r a t e s p e c i e s . In g e n e r a l , genotypic r a t i o s conformed to Hardy-Heinberg e x p e c t a t i o n s , and a l l e l e f r e q u e n c i e s w i t h i n p o p u l a t i o n s d i d not s h i f t over s h o r t time p e r i o d s . , Gene f r e q u e n c i e s d i d not vary among s t i c k l e b a c k s of d i f f e r e n t s i z e s nor among those caught by d i f f e r e n t methods from the same p o p u l a t i o n . Gene f r e q u e n c i e s at a l l polymorphic l o c i were s i g n i f i c a n t l y heterogeneous among s t i c k l e b a c k p o p u l a t i o n s . , Average f r e q u e n c i e s a t two l o c i {Pgm and Mdh-3) d i f f e r e d s i g n i f i c a n t l y between marine and freshwater f i s h . I n a d d i t i o n , average f r e q u e n c i e s at two other l o c i (Mdh-1 and Ck) were d i f f e r e n t among s t i c k l e b a c k s i n h a b i t i n g d i f f e r e n t types of freshwater environments.,Among freshwater p o p u l a t i o n s the C k 8 S and Pgm 9 0 a l l e l e s d i s p l a y e d c l i n a l geographic v a r i a b i l i t y i n frequency, p o s s i b l y as a r e s u l t of the d i f f e r e n t i a l sampling of v a r i o u s freshwater h a b i t a t types i n d i f f e r e n t r e g i o n s . A l l e l e f r e q u e n c i e s a t three l o c i (Pgm, Ck and Pgi-2) d i f f e r e d between marine s t i c k l e b a c k s c o l l e c t e d from t h e S t r a i t o f Georgia and those from waters o f f the vest c o a s t of Vancouver I s l a n d . , C a l c u l a t i o n o f Nei»s g e n e t i c d i s t a n c e i n d i c a t e d t h a t * h i l e marine p o p u l a t i o n s a r e r e l a t i v e l y homogeneous a t e l e c t r o p h o r e t i c l o c i , freshwater p o p u l a t i o n s are h i g h l y heterogeneous. The average g e n e t i c d i s t a n c e between marine and freshwater p o p u l a t i o n s was s i m i l a r t o t h a t s e p a r a t i n g p a i r s of freshwater p o p u l a t i o n s . The g e n e t i c d i s t a n c e between freshwater p o p u l a t i o n s was g r e a t e r betseen than w i t h i n watersheds, but a l l e l e d i s t r i b u t i o n s at i n d i v i d u a l l o c i d i d not d i f f e r s i g n i f i c a n t l y between two watersheds. l e v e l s o f polymorphism and h e t e r o z y g o s i t y were r e l a t i v e l y high i n p o p u l a t i o n s from the ocean, l a r g e l a k e s and l o w - l y i n g streams, and low i n those from s m a l l l a k e s and i s o l a t e d streams. Both d e t e r m i n i s t i c ( n a t u r a l s e l e c t i o n ) and s t o c h a s t i c (founder e f f e c t s and g e n e t i c d r i f t ) mechanisms can be invoked t o e x p l a i n these p a t t e r n s . , M o r p h o l o g i c a l l y and e c o l o g i c a l l y d i s t i n c t b e n t h i c and l i m n e t i c s t i c k l e b a c k s w i t h i n s i n g l e l a k e s , and freshwater and i v marine s t i c k l e b a c k s w i t h i n a stream, c o n s t i t u t e d g e n e t i c a l l y d i s c r e t e p o p u l a t i o n s . There was a s t r i k i n g congruence i n the p a t t e r n s of morphological and 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 among the p o p u l a t i o n s comprising such 'species p a i r s ' . The r e s u l t s of t h i s study are compatible with the su g g e s t i o n t h a t freshwater p o p u l a t i o n s of the study area are p o l y p h y l e t i c , and have a r i s e n independently from marine s t i c k l e b a c k s which invaded the r e g i o n s i n c e the l a s t i c e age, about 10,000 years ago. ¥ TABLE OF CONTENTS Ab s t r a c t . . . . . ....... . .............. .............. . n Table of contents ......................................... v L i s t o f t a b l e s ......................,.......-.,-.•.«.-.--. ? i i L i s t of f i g u r e s . ...................... .... ......« , • . . . . . v i i i Acknowledgments • ...• .*........•• ..................... ..... i x I n t r o d u c t i o n ........ .... ............................ • .-»«•-•-, 1 M a t e r i a l s and Methods ......................................, 6 Nomenclature .......................... *............. . 6 F i e l d C o l l e c t i o n s . . . . . . . . . . ... . . . » »•> . • ... . . • . . . . . . . 8 Cro s s i n g Techniques ................................. 9 Sample P r e p a r a t i o n . , - y . v . * . . . . 11 Gel Preparation ............................. 9....... 12 B u f f e r Systems ........................... 12 E l e c t r o p h o r e s i s ....................... . .. ... . 13 Enzyme Assay .................. ................ 14 S e c t i o n I. D e s c r i p t i o n and I n h e r i t a n c e of G a s t e r o s t e u s Isozyme P a t t e r n s ....................................... 16 C r e a t i n e Kinase . ... ......................... ........ 17 Malate Dehydrogenase ................................ 21 Phosphoglucoisomerase . . . . . . . . . . . . . . . . . . . . . . .... , 30 Phosphoglucomutase .................................. , 35 L a c t a t e Dehydrogenase ............................... 39 I s o c i t r a t e Dehydrogenase ............................ 41 S e c t i o n I I . G e n e t i c R e l a t i o n s h i p s i n Gasterosteus a c u l e a t u s .. ................ . 47 JResults ..................................... ...... . - . . 47 v i G enetic V a r i a b i l i t y w i t h i n P o p u l a t i o n s ................. 47 Polymorphism ............ .....................-•>....... 49 Het e r o z y g o s i t y ...................................., 50 Hardy-Weinberg E q u i l i b r i u m .......................... 51 M u l t i p l e Samples ................... ... 54 Genetic V a r i a b i l i t y among P o p u l a t i o n s 63 Comparison of Freshwater and Marine Po p u l a t i o n s ..... 64 Geographic V a r i a b i l i t y i n A l l e l e Frequencies 66 Comparisons of A l l e l e F r e q u e n c i e s among H a b i t a t s .... 69 Comparisons of Genetic V a r i a b i l i t y among H a b i t a t s ... 70 Comparisons Within and Between Drainage Systems ..... 72 R e l a t i o n s h i p s between Benthic and L i m n e t i c S t i c k l e b a c k s ..................................... 76 D i s c u s s i o n . . . . . .... .... . ... ....... . . . ......... ..w. .*». 80 Summary .» .... . . » . . . . ...-. ....... .•»«. .:,:...».,-. .. ••%>•*••/10.5 L i t e r a t u r e C i t e d . . , , , . . . . . . . . w . . . . - v . . . . . . . . 1 0 8 A ppendix I •...........................,,.,......>•,%>.».-. 121 Appendix I I . » . .. .... ...... . ................. • . *.... «i,>-12 4 Appendix I I I ........ ..................... ... .......... .... 126 Appendix IV .....,.,............ ............. ........ 128 Appendix V ....... ... , ................ .v. . . .. . . . .w . . ..v. .130 Appendix VI . ...... ...... . . „. .. ,..v» ,-132 Appendix VII 134 Appendix V I I I ........ .......... . ... ..... . . 13 6 v i i LIST OF TABLES Table 1. The enzymes, b u f f e r systems and s t a i n i n g s o l u t i o n s used i n t h i s study. .....................«.••,,»„••••,• ,,•*,••»,>..•.. 6 Table 2. I n h e r i t a n c e of Ck a l l e l e s i n L i t t l e Campbell R i v e r t r a c h u r u s and l e i u r u s s t i c k l e b a c k s . , . . . . , . . , . , . , . . . . . , ! . . 20 Table 3. I n h e r i t a n c e o f Mdh-1 a l l e l e s i n L i t t l e Campbell R i v e r t r a c h u r u s and l e i u r u s s t i c k l e b a c k s . ,, ,,»,,.. • .. 26 Table 4. I n h e r i t a n c e o f Mdh-3 a l l e l e s i n L i t t l e Campbell R i v e r t r a c h u r u s and l e i u r u s s t i c k l e b a c k s . . . . . , 27 Tab l e 5. I n h e r i t a n c e of Pgi-2 a l l e l e s i n L i t t l e Campbell B i v e r t r a c h u r u s and l e i u r u s s t i c k l e b a c k s . , . . . . . . . . ^ . i . . . 34 Table 6. I n h e r i t a n c e o f Pgm a l l e l e s i n L i t t l e Campbell B i v e r t r a c h u r u s and l e i u r u s s t i c k l e b a c k s . .............. 38 Table 7. Sex r a t i o s i n l a b - r e a r e d broods of L i t t l e Campbell B i v e r t r a c h u r u s and l e i u r u s s t i c k l e b a c k s as determined by IDH e l e c t r o p h o r e t i c p a t t e r n s . ....................... 43 Tab l e 8. Summary of i n t r a p o p u l a t i o a g e n e t i c v a r i a b i l i t y i n Gasterosteus a c u l e a t u s . ,-. .... ........................... 48 Table 9. A l l e l e frequency v a r i a b i l i t y i n m u l t i p l e samples from Gasterosteus p o p u l a t i o n s . . 57 Table 10. A l l e l e frequency v a r i a b i l i t y among G. a c u l e a t u s p o p u l a t i o n s . , . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . 65 Table 11. A l l e l e frequency v a r i a b i l i t y w i t h i n and between the Bear and Somass watersheds. 74 Tab l e 12. A l l e l e frequency v a r i a b i l i t y between b e n t h i c and l i m n e t i c s t i c k l e b a c k s . ................w, 77 v i i i L I S T OF FIGD8ES F i g u r e 1. T h r e e s p i n e s t i c k l e b a c k CK i s o z y m e s on a g e l s t a i n e d w i t h a g e n e r a l p r o t e i n d y e . . . . . . . . . . . . . . . . . . . . . . 17 F i g u r e 2. The- MDH i s o z y m e b a n d i n g p a t t e r n s o f t h r e e s p i n e s t i c k l e b a c k s . 22 F i g u r e 3. The t h r e e s p i n e s t i c k l e b a c k PGI i sozyme b a n d i n g pa t t e r n s . 31 F i g u r e 4. The PGM i s o z y m e b a n d i n g p a t t e r n s of t h r e e s p i n e s t i c k l e b a c k s . .. 37 F i g u r e 5... T h r e e s p i n e s t i c k l e b a c k IDH i s o z y m e b a n d i n g p a t t e r n s . , 4 2 F i g u r e 6. A l l e l e f r e q u e n c y d i f f e r e n t i a t i o n between b e n t h i c and l i m n e t i c s t i c k l e b a c k s . 79 i x ACKNOWLEDGMENTS I wish t o express s i n c e r e g r a t i t u d e to Dr. J . D. McPhail f o r the i n v a l u a b l e a s s i s t a n c e , advice and encouragememt he provided throughout the course o f t h i s study. H e a r t f e l t thanks go a l s o t o E r i c P a r k i n s o n f o r h i s t h o u g h t f u l c o n t r i b u t i o n s i n the l a b and during data a n a l y s i s , and t o Clyde Murray, T e r r y Beacham, Pe t e r W i t h l e r and others who helped with f i e l d c o l l e c t i o n s . During the study, I r e c e i v e d support i n t h e form of N. B. C. Postgraduate S c h o l a r s h i p s and a 0 . , B. C. Ki l l a m F e l l o w s h i p . 1 INTRODUCTION Taxonomic r e l a t i o n s h i p s among p o p u l a t i o n s of the t h r e e s p i n e s t i c k l e b a c k (Gasterosteas aculeatus) on the P a c i f i c coast of North America have been the s u b j e c t of l i v e l y debate, and even d i s p u t e (Heuts 1947, Hagen 1967, H i l l e r and Hubbs 1969, Hagen and McPhail 1970, B e l l 1976).,The s p e c i e s e x h i b i t s two b a s i c l i f e h i s t o r y p a t t e r n s . One form of G a s t e r o s t e u s . commonly c a l l e d t r a c h u r u s , i s an anadromous marine f i s h . t h a t breeds i n freshwater d u r i n g t h e s p r i n g , and the o t h e r , known as l e i u r u s , i s a permanent freshwater r e s i d e n t of c o a s t a l l a k e s and r i v e r s . M o r p h 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 the s p e c i e s or " s p e c i e s -complex" (McPhail and Lindsey 1970), e s p e c i a l l y among freshwater p o p u l a t i o n s , i s extreme. C h a r a c t e r s such as l a t e r a l p l a t e development and number, d o r s a l spine number and l e n g t h , g i l l r a k e r number and l e n g t h , v e n t r a l spine and p e l v i c g i r d l e development, v e r t e b r a e number, e t c . vary g r e a t l y , even over d i s t a n c e s as s h o r t as s e v e r a l hundred yards (Hagen 1967, Narver 1969, M i l l e r and Hubbs 1969, Hagen and G i l b e r t s o n 1972, Hoodie 1972a, Larsen 1976). Since many of these c h a r a c t e r s have a g e n e t i c b a s i s (Hagen 1973, Ross 1973, Hagen and G i l b e r t s o n 1973a, Avise 1976) and respond r a p i d l y to l o c a l agents of n a t u r a l s e l e c t i o n (McPhail 1969, Hagen and G i l b e r t s o n 1973b, Kynard 1972, Hoodie 1972b, Hoodie et a l . . 1973, Hoodie and Beimchen 1976, B e l l and Haglund 1978, Gross 1978) , they are of l i t t l e use as taxonomic c r i t e r i a . The purpose of t h e present study was t o examine e l e c t r o p b o r e t i c a l l y - d e t e c t a b l e genetic v a r i a b i l i t y i n enzymatic gene f r e q u e n c i e s , and i n t r e p r e t the 2 f i n d i n g s i n view o f present concepts of s t i c k l e b a c k e v o l u t i o n along the P a c i f i c c o a s t . F o s s i l records f o r C a s t e r o s t e u s on the P a c i f i c c o a s t of North America date back ten m i l l i o n years and r e v e a l that d i s t i n c t l y d i f f e r e n t freshwater and marine forms e x i s t e d at l e a s t as e a r l y as the Late Pliocene ( B e l l 1976). The present range of the t h r e e s p i n e s t i c k l e b a c k extends from A l a s k a to Baja C a l i f o r n i a (McPhail and L i n d s e y 1970). Over t h i s range the t r a c h u r u s form appears to be absent from the extreme southern areas and the l e i u r u s form from the extreme n o r t h e r n areas. Presumably, p o p u l a t i o n s along the B r i t i s h Columbia c o a s t were e s t a b l i s h e d about 10,000 years ago, a f t e r the r e t r e a t of the l a s t (Wisconsin) i c e sheet. McPhail and Lindsey (1970) suggest t h a t freshwater and marine r e f u g i a f o r G. a c u l e a t n s d u r i n g the i c e age e x i s t e d to the the south (the P a c i f i c refuge) and p o s s i b l y to the n o r t h (the Bering r e f u g e ) . S t i c k l e b a c k p o p u l a t i o n s i n h a b i t i n g the a r e a encompassed by the present study, the lower F r a s e r R i v e r V a l l e y , Vancouver I s l a n d and the S e c h e l t P e n i n s u l a , are the r e s u l t of p o s t - g l a c i a l d i s p e r s a l from the southern, and p o s s i b l y the n o r t h e r n , r e f u g i a . The wide c o a s t a l d i s t r i b u t i o n of G a s t e r o s t e u s and i t s absence from i n t e r i o r areas i n d i c a t e t h a t p o s t - g l a c i a l i n v a s i o n of the North P a c i f i c r e g i o n took p l a c e through the ocean. D i s p e r s a l along the B r i t i s h Columbia coast was probably p o s s i b l e about 9000 years ago (McPhail and L i n d s e y 1970) . S i n c e t h e l e i u r u s form has a low s a l t w a t e r t o l e r a n c e (Heuts 1947, Hootton 1976) i t i s u n l i k e l y to d i s p e r s e through t h e sea. T h e r e f o r e , the d i s p e r s i n g and c o l o n i z i n g s t i c k l e b a c k s must have been t r a c h u r u s . 3 B e l l (1976) s t a t e s t h a t temperature f l u c t u a t i o n s a t t h i s time probably caused a number of north-south s h i f t s i n the range of t r a c h u r u s along the co a s t . He suggests t h a t freshwater l e i u r u s p o p u l a t i o n s evolved from s m a l l numbers of t r a c h u r u s which invaded freshwater and then became i s o l a t e d when marine c o n d i t i o n s became unfavourable. Subsequent divergence, even i n the f a c e of probable secondary contact with marine p o p u l a t i o n s , took p l a c e i n these freshwater systems. T h i s sequence gave r i s e t o the present h i g h l y v a r i a b l e l e i u r u s phenotypes. T h i s model e n t a i l s the p o l y p h y l e t i c e v o l u t i o n of freshwater s t i c k l e b a c k s from the marine form. Because t h i s e v o l u t i o n occurred independently i n d i f f e r e n t freshwater systems, morphological s i m i l a r i t i e s among freshwater p o p u l a t i o n s i n d i f f e r e n t drainages are as l i k e l y to i n d i c a t e s i m i l a r s e l e c t i v e regimes .as common a n c e s t r y . Based on t h i s e v o l u t i o n a r y scheme, a number of hypotheses can be generated about g e n e t i c r e l a t i o n s h i p s w i t h i n and among s t i c k l e b a c k p o p u l a t i o n s . Making the assumption t h a t genetic v a r i a b i l i t y a t the enzyme l e v e l i s l e s s s u b j e c t t o n a t u r a l s e l e c t i o n , and t h e r e f o r e more time-dependent, than are morphological t r a i t s . B e l l (1976) made a s e r i e s of p r e d i c t i o n s concerning the p a t t e r n s o f 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 t h a t should be r e v e a l e d . He suggested that marine p o p u l a t i o n s should d i s p l a y g r e a t e r i n t r a popula t i o n g e n e t i c v a r i a b i l i t y than freshwater ones because, a c c o r d i n g t o the model, they are the o l d e s t i n the r e g i o n , and have had the l o n g e s t time t o ac g u i r e v a r i a b i l i t y through mutation. I n a d d i t i o n , the conti n u o u s marine environment f a c i l i t a t e s gene flow, and t h e r e f o r e the spread of 4 newly generated g e n e t i c v a r i a t i o n , freshwater p o p u l a t i o n s , on the o t h e r hand, should e x h i b i t decreased v a r i a b i l i t y owing t o founder e f f e c t s (the r e s u l t of o r i g i n a t i n g from s m a l l numbers of trac h u r u s that perhaps possessed only some f r a c t i o n of the t o t a l s p e c i e s v a r i a b i l i t y ) , and subsequent s t o c h a s t i c events t h a t o c c u r r e d when p o p u l a t i o n s i z e s were s m a l l ( g e n e t i c d r i f t , e s p e c i a l l y d u r i n g p o p u l a t i o n b o t t l e n e c k s ) . At the same time, the gr e a t e r o p p o r t u n i t y f o r gene flow among marine p o p u l a t i o n s should r e s u l t i n lower i n t e r p o p u l a t i o n g e n e t i c h e t e r o g e n e i t y among t r a c h u r u s than l e i u r u s p o p u l a t i o n s . T h i s i s because the r e s t r i c t i o n o f gene flow between l e i u r u s p o p u l a t i o n s prevents the exchange of g e n e t i c i n f o r m a t i o n . , I f freshwater s t i c k l e b a c k s a l l descended from marine forms, and s e l e c t i o n has not changed a l l e l e f r e q u e n c i e s o r e s t a b l i s h e d new a l l e l e s i n l e i u r u s p o p u l a t i o n s , then g e n e t i c l o c i t h a t a re monomorphic i n t r a c h u r u s p o p u l a t i o n s should a l s o be monomorphic i n l e i u r u s p o p u l a t i o n s ( B e l l 1976). In c o n t r a s t , l o c i t h a t are polymorphic i n tra c h u r u s p o p u l a t i o n s may or may not be polymorphic i n l e i u r u s p o p u l a t i o n s , but average f r e q u e n c i e s s h o u l d not d i f f e r between the two forms. I f f i x a t i o n i n fres h w a t e r p o p u l a t i o n s occurs a t l o c i t h a t a re polymorphic i n marine p o p u l a t i o n s , then the number of freshwater p o p u l a t i o n s f i x e d f o r each a l l e l e of a l o c u s should be p r o p o r t i o n a l to the frequency of that a l l e l e i n marine p o p u l a t i o n s . F i n a l l y , e c o l o g i c a l l y and m o r p h o l o g i c a l l y d i s s i m i l a r p o p u l a t i o n s w i t h i n a freshwater drainage system should d i s p l a y g r e a t e r 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 (due t o t h e i r common o r i g i n ) than m o r p h o l o g i c a l l y s i m i l a r p o p u l a t i o n s from d i f f e r e n t drainage 5 systems t h a t i n h a b i t s i m i l a r types of h a b i t a t s , but presumably ev o l v e d independently from the marine form.. The purpose of the present study was t o t e s t the above p r e d i c t i o n s by measuring the molecular g e n e t i c v a r i a b i l i t y i n a number of marine and freshwater p o p u l a t i o n s of Q. a c u l e a t u s . I n a d d i t i o n ; an attempt was made to t e s t the assumption of s e l e c t i v e n e u t r a l i t y , or n e a r - n e u t r a l i t y , of the observed 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 by seeking gene-environment r e l a t i o n s h i p s among p o p u l a t i o n s and gene-morphology r e l a t i o n s h i p s among c e r t a i n groups of s t i c k l e b a c k s , and by comparing r a t e s cf morphological and e l e c t r o p h o r e t i c change i n an i n t r o d u c e d s t i c k l e b a c k p o p u l a t i o n . S e c t i o n I of the r e p o r t c o n t a i n s the combined r e s u l t s and d i s c u s s i o n of two se t s of breeding experiments conducted t o e s t a b l i s h the g e n e t i c b a s i s f o r , and i n h e r i t a n c e p a t t e r n s o f , the observed 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 . S e c t i o n I I c o n s i s t s o f the r e s u l t s and d i s c u s s i o n , presented s e p a r a t e l y , of measurements of g e n e t i c h e t e r o g e n e i t y w i t h i n and among the G. a c u l e a t u s p o p u l a t i o n s examined. 6 MATERIALS AND METHODS Nomenclature The enzymes examined i n t h i s study, t h e i r I n t e r n a t i o n a l Onion o f Biochemistry (1965) numbers, and t h e i r a b b r e v i a t i o n s are l i s t e d i n Table 1. Throughout the t e x t the c a p i t a l i z e d enzyme a b b r e v i a t i o n (e.g. PGM) d e s i g n a t e s the enzyme i t s e l f , and the same a b b r e v i a t i o n w i t h only t he f i r s t l e t t e r c a p i t a l i z e d i n d i c a t e s the gene, o r l o c u s , coding f o r the enzyme. I n v a case of m u l t i p l e l o c i coding f o r d i f f e r e n t forms of a s i n g l e enzyme, the d i f f e r e n t enzymatic forms and the genes coding f o r them are d i s t i n g u i s h e d by appending a d i f f e r e n t numeral to each (e.g. MDH-1, Mdh-1). The numeric d e s i g n a t i o n of m u l t i p l e enzymatic forms i n c r e a s e s with decreasing anodal m o b i l i t y . The d i f f e r e n t a l l e l e s of a s i n g l e l o c u s a r e d i s t i n g u i s h e d by s u p e r s c r i p t numbers., A common a l l e l e o f each gene i s designated by a s u p e r s c r i p t 100 (e.g. Ck*oo) # other a l l e l e s by s u p e r s c r i p t numbers i n d i c a t i v e o f the anodal m o b i l i t y of t h e i r enzyme products r e l a t i v e to t h a t of a l l e l e 100. F i e l d C o l l e c t i o n s The s t i c k l e b a c k s used f o r e l e c t r o p h o r e s i s i n t h i s study were c o l l e c t e d from s i t e s on Vancouver I s l a n d , the S e c h e l t P e n i n s u l a and the lower F r a s e r R i v e r V a l l e y i n B r i t i s h Columbia. In a d d i t i o n , s e v e r a l s i t e s i n northwestern Washington were 7 Table 1. The enzymes, b u f f e r systems and s t a i n i n g s o l u t i o n s used i n t h i s study. Enzymes are numbered i n accordance with the I n t e r n a t i o n a l Union of Biochemistry (1965). S t a i n s f o r PGI, PGM, LBH and IDH are mixed i n 100 ml of 50 mil T r i s a d j u s t e d to pfl 7.1 with B C l . The s t a i n f o r MDH i s mixed i n 100 ml of g e l B u f f e r I . Enzyme A b b r e v i a t i o n B u f f e r S t a i n Components C r e a t i n e CK I General p r o t e i n s t a i n : Kinase 0.1% Amido Black 103 and 0. IS Napthol Blue (2.7.3.2) Black i n a 1:4:5 mix-ture o f a c e t i c a c i d ; methanol: water. „. Des t a i n : 1:4:5 a c e t i c a c i d : methanol: water. I s o c i t r a t e IDH I I or I l l 50 mg D L - N a - i s o c i t r a t e Dehydrogenase (NADP) 10 mg NADP 15 mg NBT (or MTT) (1. 1. 1.42) 5 mg PMS -50 mg MgCl 2 L a c t a t e LDH I 20 ml 0.5 M DL-Na-Dehydrogenase l a c t a t e 10 mg NAD (1.1.1.27) 15 mg NBT (or ATT) 5 mg PMS Malate MDH I I or I I I 20 ml 0.5 H DL-tfa-Dehydrogenase malate 10 rag NAD (1.1. 1. 37) 15 mg NBT (or JJTT) 5 mg PMS Phosphogluco- PGI I 50 mg Na-fructose 6-isomerase phosphate 100 u n i t s G6PDH , (5.3. 1.9) 10 mg NADP 50 mg MgCl 2 15 mg NBT (or MTT) 5 mg PMS Ehosphogluco- PGM I 200 mg K-glucos«s-1-mutase phosphate 100 u n i t s G6PDH (2.7.5. 1} 10 mg NADP • : 50 mg MgCl 2 15 mg NBT (or MTT) 5 mg PMS G6PDH Glucose-6-phosphate dehydrogenase NAD Nicotinamide adenine d i n u c l e o t i d e NADP Nicotinamide adenine d i n u c l e o t i d e phosphate N£I p - N i t r o t e t r a z o l i u m blue MTT 3-(4,5-dimethyl T h i a z o l y l - 2 ) - 2 , 5 - d i p h e n y l ,, t e t r a z o l i u m bromide EMS Phenazine methosulfate 9 sampled, A complete l i s t of the c o l l e c t i o n s i t e s and t h e i r a ssigned a b b r e v i a t i o n s , date (s) sampled and l o n g i t u d i n a l and l a t i t u d i n a l c o - o r d i n a t e s are giv e n i n Appendix I . The 79 s i t e s sampled i n c l u d e l a k e s , streams, swamps and marine h a b i t a t s . An attempt was made to c o l l e c t at l e a s t 40 f i s h from each s i t e , but t h i s was not always p o s s i b l e (Table 8)., O s u a l l y , minnow t r a p s , a long-handled d i p - n e t or a pole s e i n e (a 1.5 by 1 m n e t , with 0.6 cm mesh, supported and drawn through the water by two 2 m poles) were used f o r capture; however, s t i c k l e b a c k s were a l s o c o l l e c t e d i n 15.5 or 31 m beach s e i n e s and, from the ocean, i n an o t t e r t r a w l . Captured f i s h were bagged i n water and immediately f r o z e n on dry i c e . I n the l a b o r a t o r y , samples were s t o r e d a t -20C u n t i l used.,The enzymes examined r e t a i n e d a c t i v i t y f o r time p e r i o d s up t o and exceeding one year., However, i n most cases, samples were e l e c t r o p h o r e s e d w i t h i n f o u r months of c o l l e c t i o n . C r o s s i n g Techniques In June 1977 male and female t r a c h u r u s i n breedinq c o n d i t i o n were s e i n e d from r e g i o n s 2 km upstream from the mouth of the L i t t l e Campbell B i v e r and t r a n s p o r t e d to the l a b o r a t o r y . Crosses were made by using t e s t e s e x c i s e d from males and minced i n water t o f e r t i l i z e eggs that had been g e n t l y extruded from g r a v i d females.... To maximize the number of p a r e n t a l g e n e t i c combinations, m i l t from a s i n g l e male was sometimes used t o f e r t i l i z e egg c l u t c h e s from two d i f f e r e n t females and, s i m i l a r l y , an egg c l u t c h from one female was sometimes d i v i d e d 10 i n h a l f and f e r t i l i z e d with m i l t from d i f f e r e n t males. A l l f i s h employed as parents sere f r o z e n f o r subsequent e l e c t r o p h o r e s i s . Eggs and newly-hatched young were incubated i n i t i a l l y i n 682 ml (24 oz) g l a s s j a r s of oxygenated f r e s h water h e l d at room temperature (18C-23C), Two weeks a f t e r hatching, broods were t r a n s f e r r e d to 22.7 1 ( f i v e - g a l Ion) a q u a r i a a t 23c. ,,As the f i s h grew, broods were sub d i v i d e d among more tanks to prevent crowding. The young were f e d newly-hatched b r i n e shrimp n a u p l i i IArtemius) twice d a i l y f o r the f i r s t two months and f r o z e n b r i n e shrimp t h e r e a f t e r . O f f s p r i n g of these c r o s s e s s u f f e r e d high m o r t a l i t y throughout t h e i r e x i s t e n c e ; as eggs (fungal i n f e c t i o n ) , a t h a t c h i n g , aad upon t r a n s f e r to a g u a r i a . S u r v i v o r s ( r e p r e s e n t i n g 16 of 22 o r i g i n a l crosses) were f r o z e n f o r e l e c t r o p h o r e s i s a t i n t e r v a l s from 6 weeks t o 6 months a f t e r h a t c h i n g . , B e t t e r sample s i z e s were obtained from a second s e t of c r o s s e s c a r r i e d out i n A p r i l 1979. L e i u r a s s t i c k l e b a c k s c o l l e c t e d 15 km upstream from the mouth of the L i t t l e Campbell R i v e r were employed as parents. Four males placed i n separate 22.7 1 a g u a r i a b u i l t nests and courted g r a v i d females t h a t »ere i n t r o d u c e d i n t o t h e tanks. Spawning o c c u r r e d n a t u r a l l y ; one male f e r t i l i z e d eggs from two females and the o t h e r t h r e e males each f e r t i l i z e d eggs from one female. Female parents were f r o z e n immediately a f t e r egg d e p o s i t i o n * Hales were l e f t to care f o r the eggs and young u n t i l one week a f t e r h a t c h i n g , and then removed and f r o z e n . The young were maintained at room temperature (19C-23C) on a d i e t of b r i n e shrimp n a u p l i i and, a f t e r s e v e r a l weeks, chopped 11 T u c i f e x worms, One week a f t e r emergence from the nest each brood was s u b d i v i d e d among s e v e r a l a q u a r i a . Samples of young were removed and f r o z e n f o r e l e c t r o p h o r e s i s at f o u r , e i g h t and s i x t e e n weeks a f t e r hatching,, M o r t a l i t y i n these c r o s s e s was low, and i t i s l i k e l y t h a t the f i s h so obtained represented random samples of e n t i r e broods. Sample P r e p a r a t i o n The enzymes examined i n t h i s study are a l l present i n Gast e r o s t e u s muscle t i s s u e . To a i d t i s s u e removal, s t i c k l e b a c k s f r o z e n i n water were p a r t i a l l y thawed, separated, l a i d out on a t r a y and r e f r o z e n . F o r s t i c k l e b a c k s over 2 cm i n l e n g t h , samples were s l i c e d from e i t h e r the l e f t or r i g h t s i d e of the caudal r e g i o n . The e n t i r e caudal region (both s i d e s ) was used i n f i s h between 1 and 2 cm i n l e n g t h , and the whole f i s h was used when i t s l ength was l e s s than 1 cm. When e n t i r e f i s h were used, enzymes from other t i s s u e s s t a i n e d , but these were e a s i l y d i s t i n g u i s h e d from muscle enzymatic forms.. The f r o z e n t i s s u e samples were placed i n 13 x 75 mm p l a s t i c t e s t tubes with an equal volume o f d i s t i l l e d water, and the t e s t tube rack was kept on i c e . Samples were then ground thoroughly with a g l a s s rod and r e f r o z e n . These tubes sere s t o r e d f o r up to a week p r i o r t o e l e c t r o p h o r e s i s . For use, they were c e n t r i f u g e d f o r 6 min. a t 3000 rpm, and kept on i c e while the g e l s were loaded. Gel P r e p a r a t i o n 12 The e l e c t r o p h o r e t i c equipment and techniques employed were adopted, with s l i g h t m o d i f i c a t i o n s , from those d e s c r i b e d i n d e t a i l by May (1975) . The g e l mold c o n s i s t e d of f o u r p l e x i g l a s s s t r i p s clamped to a f l a t p l e x i g l a s s sheet, 26.7 cm (10.5 in) x 17.8 cm (7 in) x 0.64 cm (0.25 in) . The s t r i p s forming the l e n g t h of the mold were 21.6 cm (8.5 in) x 1.9 cm (0.75 in) x 1.3 cm (0.5 in) and edging t h e width were 17.8 cm (7 in) x 1.9 cm (0.75 in) x 1.3 cm (0.5 in) . Gels c o n s i s t e d o f 45.5 g of E l e c t r o s t a r c h (Lot 307, Otto H i l l e r E l e c t r o s t a r c h Co., Madison, His.), i n 350 ml of b u f f e r . Approximately one-quarter of the b u f f e r was added to t h e s t a r c h i n a 500 ml f l a s k and s w i r l e d t o d i s s o l v e the s t a r c h . The remaining b u f f e r was heated t o b o i l i n g and, with constant s w i r l i n g , added to t h e f l a s k c o n t a i n i n g the s t a r c h mixture. The f l a s k contents were heated to v i g o r o u s b o i l i n g , degassed with an a s p i r a t o r f o r one minute and f i n a l l y poured i n t o a g e l mold. The g e l was ready f o r use a f t e r c o o l i n g at room temperature f o r one-h a l f hour. , B u f f e r Systems Three b u f f e r systems were used: (I) E l e c t r o d e b u f f e r : 60 mM l i t h i u m hydroxide, 300. mfl b o r i c a c i d , pH 8.1. Gel b u f f e r : 30 mM T r i s , 5mM mon©hydrate c i t r i c a c i d , pH 8.5. G e l s c o n s i s t e d of 99SE g e l b u f f e r and 1% e l e c t r o d e b u f f e r . (Ridgway e t a l . 1970) . (II) E l e c t r o d e b u f f e r : 40 mM monohydrate c i t r i c a c i d a d j u s t e d to 1 3 pH 6.1 with N- (3-Aminopropyl) -morpholine. „ G e l b u f f e r : a 1 i n 20 d i l u t i o n ( i n d i s t i l l e d water) of e l e c t r o d e b u f f e r . (Clayton and T r e t i a k 1972) . ( I l l ) E l e c t r o d e b u f f e r : 135 mfl T r i s , 45 mM monohydrate c i t r i c a c i d , pH 7.0., G e l b u f f e r : a 1 i n 15 d i l u t i o n ( i n d i s t i l l e d water) o f t h e e l e c t r o d e b u f f e r . , (Ayala e t a l . 1972). E l e c t r o p h o r e s i s For e l e c t r o p h o r e s i s , the two long p l e x i g l a s s s t r i p s were removed from the g e l form and a cut made alo n g the l e n g t h of the g e l approximately 3 cm from one exposed edge. T h i s c u t separated the g e l i n t o two p i e c e s of unegual s i z e . F i l t e r paper ( S c h l e i c h e r and S c h u e l l , grade 470) wicks, 0«2 x 1 cm i n s i z e , were dipped i n the muscle t i s s u e samples and s t a t i o n e d v e r t i c a l l y along the cut g e l s u r f a c e . At r e g u l a r i n t e r v a l s wicks dipped i n d i l u t e red food c o l o u r i n g were i n s e r t e d as markers. A t o t a l of 40 wicks, r e p r e s e n t i n g muscle samples from 40 d i f f e r e n t f i s h , were loaded on one g e l . Then the two g e l p i e c e s , with wicks sandwiched between* sere pressed f i r m l y back together and the g e l covered with p l a s t i c wrap. T h i s wrap was f o l d e d back to expose approximately 2 cm of g e l along each long edge. The p l e x i g l a s s p l a t e s u p p o r t i n g t h e g e l was placed between two b u f f e r t r a y s c o n t a i n i n g platinum wire e l e c t r o d e s . Absorbent 14 c l o t h s were used t o e s t a b l i s h c o n t a c t between the exposed g e l edges and the e l e c t r o d e b u f f e r i n the t r a y s . The e l e c t r o d e s then were connected t o a power source i n such a way t h a t the g e l edge n e a r e s t the wicks formed the c a t h o d a l (negative) end. a f t e r ten minutes of e l e c t r o p h o r e s i s the wicks were removed and the two g e l p i e c e s pressed f i r m l y t o g e t h e r . The e n t i r e g e l and b u f f e r t r a y s then were covered with another sheet o f p l a s t i c wrap and an i c e pack was placed on the g e l . . A l l of the enzymes s t u d i e d , and the food c o l o u r i n g marker used, migrated a n o d a l l y . F o r b u f f e r systems I and I I I , 200-250 v o l t s (40-60 milliamps) were a p p l i e d u n t i l the red dye had migrated to w i t h i n 1.5 cm of t h e anodal g e l edge (4-5 h o u r s ) . For b u f f e r system I I , 60 m i l l i a m p s (200-300 v o l t s ) were a p p l i e d u n t i l the r e d dye marker reached the anodal g e l edge (about 4 h o u r s ) . Enzyme Assay A f t e r e l e c t r o p h o r e s i s the remaining two p l e x i g l a s s s t r i p s were removed from the g e l form. The c a t h o d a l g e l s t r i p was d i s c a r d e d . T h i n p l e x i g l a s s s t r i p s , 2 mm t h i c k , were stacked along the two l o n g edges of t h e g e l , and used as guides along which nylon thread was drawn t o s l i c e the g e l i n t o l a y e r s . The t o p and bottom l a y e r s were d i s c a r d e d , and the remaining s l i c e s placed i n separate s t a i n i n g t r a y s . These s l i c e s were incubated at 37C i n the s p e c i f i c and g e n e r a l ( f o r CK) enzyme s t a i n s l i s t e d i n T a b l e 1. Most o f the s t a i n s are from, or adapted from, r e c i p e s of Shaw and Prasad (1970). G e l s i n s p e c i f i c s t a i n s were incubated u n t i l bands were c l e a r (up to one hour) and then 15 s c o r e d . The g e l i n the general p r o t e i n s t a i n was i n c u b a t e d f o r 20 minutes, then r i n s e d s e v e r a l times with water and l e f t i n the d e s t a i n i n g s o l u t i o n (Table 1) o v e r n i g h t . I t was scored the f o l l o w i n g day. 16 SECTION I . DESCRIPTION AND INHERITANCE OF GASTEBOSTEUS ISOZYME PATTEBNS E l e c t r o p h o r e t i c examination of v a r i a b i l i t y a t s i n g l e l o c i coding f o r enzymatic p r o t e i n s provides a simple and a c c u r a t e method of determining g e n e t i c d i f f e r e n t i a t i o n both w i t h i n and between s p e c i e s . E l e c t r o p h o r e s i s p r o v i d e s minimal e s t i m a t e s of the g e n e t i c d i s t a n c e s e p a r a t i n g p o p u l a t i o n s because o n l y about o n e - t h i r d of a l l p o s s i b l e p o i n t mutations i n DNA .nucleotide seguences w i l l produce e l e c t r o p h o r e t i c a l l y - d e t e c t a b l e changes i n the p r o t e i n products (Shaw 1965) . However, the codominant e x p r e s s i o n { i . e . l a c k o f dominance) of a l l e l e s t h a t can be d e t e c t e d enables the p r e c i s e assessment of t h e i r gene and genotype f r e q u e n c i e s . Care must be taken i n the i n t e r p r e t a t i o n of h e t e r o g e n e i t y i n isozyme p a t t e r n s . Sources of v a r i a t i o n other than simple a l l e l i c 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 c l u d e changes i n gene e x p r e s s i o n d u r i n g development, v a r i a b i l i t y due to environmental f a c t o r s such as temperature and s a l i n i t y , and i n v i t r o changes brought about by the c o n d i t i o n s of sample storage and e x t r a c t i o n procedures ( A l l e n d o r f and Utter 1979). U s u a l l y banding p a t t e r n s show t y p i c a l Hardy-Weinberg p r o p o r t i o n s and t h i s i s o f t e n c o n s i d e r e d s u f f i c i e n t evidence f o r the underlying g e n e t i c b a s i s of the v a r i a b i l i t y . , H o w e v e r , breeding s t u d i e s are r e g u i r e d f o r a b s o l u t e c o n f i r m a t i o n . T h i s s e c t i o n c o n s i s t s o f d e s c r i p t i o n of the observed G a s t e r o s t e u s isozyme p a t t e r n s and t h e i r comparison with those of other s p e c i e s . Isozyme p a t t e r n s of progeny of the L i t t l e 17 Campbell c r o s s e s are t e s t e d f o r conformation to p r e d i c t e d genotypic p r o p o r t i o n s based on a Mendelian i n t e r p r e t a t i o n of v a r i a b i l i t y i n t h e i r parents. In a l l cases, isozyme p a t t e r n s of the young resembled those of the a d u l t s . T h i s i n d i c a t e s an absence of e i t h e r o n t o g e n e t i c or environmental i n f l u e n c e s on the p a t t e r n s . The a l l e l i c i n t e r p r e t a t i o n of v a r i a b i l i t y was confirmed f o r a l l enzymes but one (IDH). C r e a t i n e Kinase Phenotypic v a r i a b i l i t y i n Gasterostens CK p a t t e r n s was scored from g e l s s t a i n e d with t h e g e n e r a l p r o t e i n s t a i n amido Black 10B ( M i c h i e l 1977) . The isozymes monitored were by f a r the darkest s t a i n i n g bands on the g e l . T h i s i s c o n s i s t e n t with the demonstrations by Gosselin-Eey et a l . (T968) and Go s s e l i n - f i e y and Gerday (1970) t h a t c r e a t i n e kinase c o n s t i t u t e s a l a r g e p r o p o r t i o n (16% i n carp) o f the white muscle c e l l p r o t e i n s i n f i s h . In a d d i t i o n , the CK banding p a t t e r n of s t i c k l e b a c k s ( F i g . 1) conforms with t h a t found i n other t e l e o s t s by s p e c i f i c s t a i n i n g methods ( F e r r i s and S h i t t 1978) . , S t i c k l e b a c k s possess e i t h e r one or both of two CK isozyme bands (A 1 a* and A 2 A 2 i n F i g . , 1) . R e s u l t s of the L i t t l e Campbell l e i u r u s c r o s s e s (Table 2) i n d i c a t e t h a t t h i s v a r i a b i l i t y i s under the c o n t r o l of a s i n g l e autosomal l o c u s with two a l l e l e s . I n d i v i d u a l s d i s p l a y i n g the A 1A* band are homozygous f o r an a l l e l e termed Ck*o° (FF i n Table 2), those d i s p l a y i n g the A 2 A 2 band are homozygous f o r an a l l e l e termed C k 8 5 (SS) and those d i s p l a y i n g both bands are C k * o ° / C k 8 5 (FS) he t e r o z y g o t e s . The 18 F i g u r e 1, Threespine s t i c k l e b a c k CK isozymes on a g e l s t a i n e d with a g e n e r a l p r o t e i n dye. I n d i v i d u a l s homozygous f o r C k 1 D ° (nos. . 1 and 3) d i s p l a y the A*A l band, those homozygous f o r C k 8 5 (no. 4) d i s p l a y t h e a 2 A 2 band, and C k * « > V C k 8 S heterozygotes (nos. 2 and 5) d i s p l a y both bands. 19 homozygous and heterozygous l e i u r u s parents of every c r o s s produced progeny d i s p l a y i n g Mendelian p r o p o r t i o n s of genotypes c o n s i s t e n t with c o n t r o l by a s i n g l e autosomal gene with two a l l e l e s . .The L i t t l e Campbell t r a c h u r u s p o p u l a t i o n i s monomorphic f o r t h e C k 9 5 a l l e l e , and a l l parents and progeny of crosses i n v o l v i n g these f i s h d i s p l a y e d only the A 2A 2 band (Table 2). C r e a t i n e k i n a s e i s a dimeric enzyme (Dawson et a l . 1967, Gosselin-Rey and Gerday 1970} and, as such, should e x i s t i n three isozyme forms i n i n d i v i d u a l s heterozygous at a Ck l o c u s . The expected banding p a t t e r n based on a binomial a s s o c i a t i o n of the two p e p t i d e s produced c o n s i s t s of a 1:2:1 r a t i o of horaodimerrheterodimer:homodimer. In v e r t e b r a t e s other than f i s h , h eterozygotes f o r muscle c r e a t i n e kinase (commonly r e f e r r e d t o as CK-A) e x h i b i t j u s t such a 3-banded p a t t e r n ( F e r r i s and B h i t t 1978) . However, as f i r s t noted by Scopes and Gosselin-Bey (1968) f o r carp (C y p r i n us c a r p i o ) and T i l a p i a spp., t e l e o s t s heterozygous f o r muscle c r e a t i n e k i n a s e t y p i c a l l y possess only two e l e c t r o p h o r e t i c a l l y d i s t i n c t isozyme forms, and consequently d i s p l a y a 2-banded p a t t e r n s i m i l a r t o that of Gasterosteus. , F e r r i s and Whitt (1978) extended the examination of CK-A phenotypes to s p e c i e s belonging to seven orders of bony f i s h e s and found the 2-banded p a t t e r n to be c h a r a c t e r i s t i c of i n d i v i d u a l s heterozygous at any one Ck-A l o c u s i n a l l s p e c i e s t h a t showed ge n e t i c v a r i a b i l i t y . They demonstrated t h a t muscle CK of f i s h e s e x i s t s o n l y i n homodimeric forms; i n heterozygotes t h e expected heterodimer of i n t e r m e d i a t e m o b i l i t y i s not formed. . T h e i r f i n d i n g s i n d i c a t e d t h a t t h e r e e x i s t s a " s p a t i a l and/or temporal i s o l a t i o n of the s y n t h e s i s and/or assembly" of c r e a t i n e 20 Table 2. I n h e r i t a n c e o f ck a l l e l e s i n L i t t l e Campoell Hiver t r a c h u r u s and l e i u r u s s t i c k l e b a c k s . I n both s e t s of c r o s s e s the f a s t e r a l l e l e (F) i s Ck»oo a n a the slower a l l e l e (s) i s C k 8 S . P a r e n t a l genotypes i n b r a c k e t s were i n f e r r e d from progeny genotypic r a t i o s . , . P a r e n t a l No. o f Progeny Genotypes Crosses FF FS SS Trachurus or* SS ? SS L e i u r u s d* FF ? FS 13 0 0 198 1 30 28 0 o* FF ? FS 19 27 0 o* FS ? FS 20 36 19 P a r e n t a l No. o f Progeny Genotypes Crosses E£ FS SS o* (SS) ? (SS) 0 0 49 cf FS ? FS 7 21 13 o* SS ? FS 0 26 32 21 kinase p e p t i d e s coded f o r by the two Ck a l l e l e s l o c a t e d on homologous chromosomes i n muscle c e l l s . , The e v o l u t i o n a r y s i g n i f i c a n c e of t h i s widespread r e s t r i c t i o n of subunit i n t e r a c t i o n i n the muscle CK of bony f i s h e s i s not known ( F e r r i s and Whitt 1978). Malate Dehydrogenase The r e l a t i v e l y complex p a t t e r n of Gasterosteus MDB ( F i g . . 2) i s c o n s i s t e n t with t h e presence, i n v e r t e b r a t e s , of g e n e t i c a l l y d i s t i n c t c y t o p l a s m i c (supernatant) and m i t o c h o n d r i a l malate dehydrogenases (Thorne e t a l . 1963, Davidson and Cortner 1967, Wheat and Whitt 1971)., Moreover, many organisms, i n c l u d i n g numerous t e l e o s t s p e c i e s , possess two or more genes coding f o r supernatant malate dehydrogenases of d i f f e r i n g e l e c t r o p h o r e t i c m o b i l i t i e s ( B a i l e y et a l . , / 1970, Whitt 1970, Wheat and Whitt 1971, Clayton et a l . , 1973). G e n e t i c a l l y d i s t i n c t m u l t i p l e m i t o c h o n d r i a l forms may a l s o e x i s t i n some s p e c i e s ( K i t t o et a l . 1966). . MDH i s a dimer, and the a s s o c i a t i o n of p o l y p e p t i d e products o f d i f f e r e n t a l l e l e s of a s i n g l e g e n e t i c l o c u s , or two d i f f e r e n t l o c i , t o form f u n c t i o n a l h e t e r o d i m e r i c enzymes r e s u l t s i n the p r o d u c t i o n of numerous isozymes. The d i m e r i z a t i o n of peptides coded f o r by s e p a r a t e supernatant l o c i i s common ( B a i l e y e t a l . 1970, Clayton et a l . 1971), and i n v i t r o techniques such as f r e e z i n g and thawing t i s s u e enhance the a s s o c i a t i o n of peptides coded f o r by supernatant and m i t o c h o n d r i a l l o c i w i t h i n a s p e c i e s ( C h i l s o n e t a l . , 1966), or two supernatant or m i t o c h o n d r i a l 22 F i g u r e 2. The MDH isozyme banding p a t t e r n s of t h r e e s p i n e s t i c k l e b a c k s . . I n d i v i d u a l s homozygous at the Mdh-1 l o c u s f o r the Mdh-1 1 0 0 a l l e l e (nos. 2, 4 and 6) possess the A 1 A 1 band, those homozygous f o r Mdh-1 8 2 (no. 3} possess the A 2A 2 band, and Mdh-1* 0 0/ffldh-1 8 2 h e t e r o z y g o t e s (nos. 1 and 5) possess a l l three A*A», A*A 2 and A 2 A 2 bands. I n d i v i d u a l s homozygous at t h e Mdh-3 locus f o r 8 d h - 3 1 0 0 (nos.. 1 and 4) possess the M*M* and M*X bands, Mdh-3 S S homozygotes (nos. 3 and 5) possess t h e M 2M 2 and H 2X bands, and Mdh-3*°°/Hdh-3 5 S h e t e r o z y g o t e s (nos. 2 and 6) possess M1M.1, H 2H 2, M»X and fi2X. A l l s t i c k l e b a c k s possess the BB band, p o s s i b l y the homodimeric product of a monomorphic Hdh-2 l o c u s . The A*B and A 2B bands r e p r e s e n t h y b r i d isozymes. MDH 1 2 3 4 5 6 23 p e p t i d e s from d i f f e r e n t s p e c i e s (ChiIson e t a l . 1966, Sheat and Whitt 1971, C l a y t o n e t a l . 1973). The HDH p a t t e r n of Gast e r o s t e u s ( P i g . ,2) i s best explained i n terms of three s t r u c t u r a l l o c i , termed Mdh-1, Mdh-2 and Mdh-3 i n order o f de c r e a s i n g m o b i l i t y of enzyme products. The most anodal (Mdh-1) and most c a t h o d a l (Mdh-3) of the l o c i a re s i g n i f i c a n t l y polymorphic, while Hdh-2 i s a p p a r e n t l y v i r t u a l l y monomorphic i n a l l p o p u l a t i o n s . I n d i v i d u a l s homozygous f o r the most common Mdh-1 a l l e l e (Mdh-1 1 0 0) possess the f a s t - m i g r a t i n g homodimeric isozyme l a b e l l e d k1Al i n F i g . 2. I n d i v i d u a l s heterozygous a t t h i s l o c u s f o r the Mdh-1 1 0 0 a l l e l e and a v a r i a n t a l l e l e Mdh-1 8 2 d i s p l a y the c h a r a c t e r i s t i c 3-banded phenotype produced by a heterozygous l o c u s coding f o r a di m e r i c enzyme. The bands l a b e l l e d A 2 A 2 and A 1 A 2 r e p r e s e n t the v a r i a n t homodimeric and the h e t e r o d i m e r i c isozymes r e s p e c t i v e l y . The r e l a t i v e i n t e n s i t y of s t a i n i n g of the A 1A 1, A 1 A 2, A 2A 2 bands i s 1:2:1 as expected, R a t i o s of progeny phenotypes i n the L i t t l e Campbell crosses support the d i a l l e l i c , s i n g l e l o c u s i n t e r p r e t a t i o n of v a r i a b i l i t y i n t h i s r e g i o n (Table 3). Crosses i n which both parents were homozygous f o r t h e Mdh-1 1 0 0 a l l e l l e (FF) produced a l l FF progeny. C r o s s e s i n which one parent o f e i t h e r sex was heterozygous f o r the Mdh-1 1 0 0 and Mdh-1 8 2 a l l e l e s (FS), and the other homozygous FF, produced one-half FF and one-half FS progeny as expected. U n f o r t u n a t e l y , the low frequency of the Mdh-1 8 2 a l l e l e i n both L i t t l e Campbell p o p u l a t i o n s p r e c l u d e d the op p o r t u n i t y o f using an Mdh-1 8 2 homozygote i n the l a b c r o s s e s , but such i n d i v i d u a l s were present i n other p o p u l a t i o n s . Their 24 e l e c t r o p h o r e t i c p a t t e r n i n the MDH-1 r e g i o n c o n s i s t e d of only the a 2 a 2 homodimeric isozyme ( F i g . 2). The bands l a b e l l e d A*B and a?B i n F i g . a 2 represent h e t e r o d i m e r i c h y b r i d isozymes formed by the a s s o c i a t i o n of pe p t i d e products of the Mdh-1 l o c u s and a second, more cathodal Bdh-2 l o c u s t o be d i s c u s s e d below. T h e i r h y b r i d nature i s i n d i c a t e d by the complete c o r r e l a t i o n of t h e i r phenotypic v a r i a b i l i t y with genotypic v a r i a t i o n a t the Hdh-1 l o c u s . Thus an M d h - 1 1 0 0 homozygote possesses o n l y one MDH-1 peptide (a*) to combine with an MDH-2 peptide (B) , and one h y b r i d isozyme (A*B) r e s u l t s . . a n Mdh -1* 0 0/Mdh -1 8 2 heterozygote possesses two MDH-1 p e p t i d e s ( A 1 and a 2) to combine with the MDH-2 p e p t i d e B, and two isozymes r e s u l t (A 1 B and A 2 B ) . an Mdh -1 8 2 homozygote possesses o n l y the a 2B h y b r i d isozyme ( F i g . 2) . Next most e a s i l y accounted f o r i n g e n e t i c and s t r u c t u r a l terms a re the catho d a l isozymes l a b e l l e d M1 M1 and M2M2. V a r i a b i l i t y i n t h i s r e g i o n was found to be under the c o n t r o l of a s i n g l e autosomal l o c u s termed Mdh-3, i n d i v i d u a l s homozygous f o r the Mdh-3 1 0 0 a l l e l e possessed the MlM* isozyme o n l y , while i n d i v i d u a l s homozygous f o r the Mdh-3 5 S a l l e l e possessed the M 2M 2 isozyme. ,Heterozygotes possessed both M 1H 1 and M 2M 2 isozyme bands. Such a two, rather than 3-banded p a t t e r n i n the heterozygote i s i n d i c a t i v e of monomeric r a t h e r than dimeric p r o t e i n s t r u c t u r e . . I t i s p o s s i b l e t h a t bands M1M* and fl2M2 are i n v i t r o d i s s o c i a t e d monomeric p e p t i d e s coded f o r by the two a l l e l e s , and t h a t i n v i v o they combine to form homo- and heterodimers. a l t e r n a t i v e l y , t h e pe p t i d e s produced by the Mdh-3 1 0 0 and Mdh-3 5 S a l l e l e s may only a s s o c i a t e t o form homodimers. 25 and a f u n c t i o n a l h e t e r o d i m e r i c isozyme of in t e r m e d i a t e m o b i l i t y i s not produced (as with CK). A f i n a l p o s s i b i l i t y i s that M1 H 1 and M 2H 2 are a c t u a l l y heterodimers, formed by the h y b r i d i z a t i o n of p e p t i d e s from a l o c u s c a t h o d a l t o the o r i g i n (and hence not observed on the gel) and a polymorphic anodal Mdh-3 l o c u s whose products c o i n c i d e with the products of the Hdh-2 l o c u s (see below)..,At any r a t e , r e s u l t s o f the L i t t l e Campbell trachurus and l e i u r u s c r o s s e s confirm t h a t t h e v a r i a b i l i t y i s due t o two a l l e l e s of a s i n g l e autosomal gene (Table 4) . A l l p o s s i b l e combinations of homozygous M d h - 3 1 0 0 (FF), homozygous H d h - 3 5 S (SS), and heterozygous Hdh - 3 1 0 0/Mdh - 3 5 5 (ps) p a r e n t a l genotypes were achieved i n these c r o s s e s , and i n every case progeny genotypes and r a t i o s conformed with Mendelian e x p e c t a t i o n s . Phenotypic v a r i a b i l i t y i n the g e l r e g i o n c o n t a i n i n g the M*X, BB and M2X bands ( F i g . 2) d i r e c t l y r e f l e c t s genotypic v a r i a b i l i t y at the Mdh-3 l o c u s . Thus, i n d i v i d u a l s homozygous f o r M d h - 3 1 0 0 and possessing the HIM1 band a l s o d i s p l a y the H 4X and BB bands. . Mdh - 3 5 5 homozygotes, with t h e M2M2 band, d i s p l a y t he M2X and BB bands. Mdh-3 heterozygotes, i n a d d i t i o n t o the H 1 M1 and H 2H 2 bands, possess a l l three H 4X, BB and H 2X bands. The r e l a t i v e s t a i n i n g i n t e n s i t y o f the 3-banded phenotype i s 1:2+: 1. The r e l a t i v e s t a i n i n g i n t e n s i t y of the BB and whichever other band possessed by a 2-banded i n d i v i d u a l i s 1+: 1. The simplest e x p l a n a t i o n f o r the bands o f t h i s r e g i o n i s that the c e n t r a l , d a r k - s t a i n i n g BB band r e p r e s e n t s the homodimeric isozyme of a t h i r d Hdh l o c u s ( M d h - 2 ) V a r i a b i l i t y at t h i s Hdh-2 l o c u s might be d i f f i c u l t t o score because of the proximity of the MJX and H 2X bands on the g e l . However, l i k e the v a r i a b i l i t y at the Mdh-1 26 Ta b l e 3. I n h e r i t a n c e o f Hdh-1 a l l e l e s i n L i t t l e Campaall Biv°r t r a c h u r u s and l e i u r u s s t i c k l e b a c k s . In both s e t s of c r o s s e s the f a s t e r a l l e l e (F) i s Mdh-1ioo and the slower a l l e l e (sj i s Mdh-1 ® 2 . P a r e n t a l genotypes i n brackets were i n f e r r e d from progeny geno t y p i c r a t i o s . P a r e n t a l Genotypes No. of Crosses Progeny FF FS SS P a r e n t a l Genotypes No. of Crosses Progeny Fir' FS SS Trachurus <f FF ? FF 11 119 0 0 ?:l <f ¥ FS FF 1 27 36 0 <f (FF) ? (FF) 3 49 0 0 ? FF FS 1 a 8 0 L e i u r u s <f FF $ FF 4 224 0 0 if-% FF FS 1 3U 29 0 27 Table 4, I n h e r i t a n c e o f Hdh-3 a l l e l e s i n L i t t l e Campbell fiiver t r a c h u r u s and l e i u r u s s t i c k l e b a c k s , I n both s e t s of c r o s s e s the f a s t e r a l l e l e (F) i s Mdh-3 l°o and the slower a l l e l e (S) i s Hdh-3 S S , P a r e n t a l genotypes i n b r a c k e t s were i n f e r r e d from progeny genotypic r a t i o s , P a r e n t a l Genotypes Progeny P a r e n t a l Genotypes Progeny Male Female FF FS SS Hale Female FF FS SS Trachurus (!) ss CD SS 0 0 16 (8) SS (10.) SS 0 0 10 <2) FS (2) FF 21 22 0 (9) FF (9) FS 3 3 0 (3) FS (3) FS 1 5 4 C?) FF (10) SS 0 11 0 (4) FS (4) SS 0 7 3 (10) SS (11) SS 0 0 10 C5) (SS) (5) (*S) 0 11 4 (10) SS (12) SS 0 0 7 (6) SS (7) SS 0 0 10 o n FS (12) SS 0 5 5 (7) FS 18) SS 0 9. 11 (12) (SS) (13) FS 0 7 3 (8) SS (9) FS 0 7 4 L e i u r u s f13) FS (15) FF 33 26 0 (15) FF (18) FF 42 o 0 f 13) FS (16) FF 26 21 0 (16) FF (19) FF 58 0 0 114) FF (17) FF 77 0 0 28 l o c u s , v a r i a b i l i t y at t h e Hdh-2 l o c u s should r e s u l t i n a d d i t i o n a l forms of hyb r i d A * B (and A 2B) isozymes. In f a c t , e x t r a bands c a t h o d a l t o both t h e BB and A*B (or A 2B) isozymes were observed i n some p o p u l a t i o n s . T h i s p u t a t i v e Mdh-2 v a r i a b i l i t y was not monitored because i t was rare and d i f f i c u l t t o i n t e r p r e t . The d i r e c t correspondence of the presence of M*X and M2X with the presence o f M lM 1 and M 2M 2 i n d i c a t e s that M 1! and M2X are isozymes c o n t a i n i n g t h e peptides coded f o r by the Mdh-3 l o c u s . One p o s s i b l e e x p l a n a t i o n i s t h a t while the M 1H 1 and M 2M 2 bands represent the monomeric peptides coded f o r by Mdh-3, M*X and M2X represent the homodiraeric a s s o c i a t i o n s of these peptides and a heterodimer of int e r m e d i a t e m o b i l i t y c o i n c i d e s with, and th e r e f o r e cannot be d i s t i n g u i s h e d from, the BB isozyme. The problem with t h i s i n t e r p r e t a t i o n i s th a t the s i e v e - l i k e p r o p e r t i e s of s t a r c h g e l sh o u l d allow the s m a l l e r monomers t o migrate more q u i c k l y , and t h e r e f o r e f a r t h e r toward t he anode, than the diraers. More l i k e l y , i f fllX and M2X a c t u a l l y are homodimeric isozymes formed by pept i d e s of the polymorphic Mdh-3 l o c u s , then H 1M 1 and M 2M 2 are heterodimers composed o f one peptide from the Mdh-3 l o c u s (M1 or M2) and one peptid e from a ca t h o d a l l o c u s , Mdh-4, t h a t i s not v i s i b l e on the g e l . Thus, v a r i a b i l i t y a t the Mdh-3 lo c u s can be measured e i t h e r by s c o r i n g v a r i a b i l i t y i n MlX and H2X or v a r i a b i l i t y i n M1M1 and M 2M 2. Gasterosteus muscle t i s s u e was not subjected to s u b c e l l u l a r f r a c t i o n a t i o n t o determine the o r i g i n ( m i t o c h o n d r i a l or cytoplasmic) of the v a r i o u s MDH isozymes., However, the c l o s e s i m i l a r i t y o f the s t i c k l e b a c k p a t t e r n t o t h a t d e s c r i b e d f o r 29 c e r t a i n other t e l e o s t s permits some t e n t a t i v e c l a s s i f i c a t i o n . In g e n e r a l , m i t o c h o n d r i a l HDH i s more c a t h o d a l than supernatant HDH ( B a i l e y e t a l . 1970, Clay t o n e t a l . 1973), although e x c e p t i o n s are known i n the k i l l i f i s h , Fundulus h e t e r o c l i t u s (Whitt 1970) and P a c i f i c y e l l o w - f i n tuna, Meothunnus macropterus ( K i t t o and Lewis 1967) . . Vertebrate m i t o c h o n d r i a l H D H i s c h a r a c t e r i z e d by i t s r e l a t i v e l y high t h e r m o l a b i l i t y ( K i t t o and Lewis 1967). Observation of mi t o c h o n d r i a l HDH a c t i v i t y i s dependent upon m i t o c h o n d r i a l r u p t u r e , and i t s a c t i v i t y i s thus reduced or absent i n t i s s u e samples not ground o r frozen p r i o r t o e l e c t r o p h o r e s i s . F i n a l l y , no m i t o c h o n d r i a l MDH can be detected by e l e c t r o p h o r e t i c techniques i n t h e l i v e r s of a t l e a s t some f i s h s p e c i e s , such as the walleye S t i z o s t e d i o n vitreum yitreum and sauger S., canadense (Clayton e t a l . , 1973) and the saury C o l o l a b i s s a i r a (Numachi 1970). The slow-migrating H 1M 1 and M 2H 2 isozymes coded f o r by th e Hdh-3 l o c u s a p p a r e n t l y f u l f i l these c r i t e r i a . They tend to be c a t h o d a l and t h e i r a c t i v i t y was reduced i n samples ground, but not f r o z e n , before e l e c t r o p h o r e s i s and i n samples i n a d e q u a t e l y cooled during e l e c t r o p h o r e s i s . Moreover, l i v e r t i s s u e samples d i d not d i s p l a y t h e H 1H 1 or H 2H 2 isoenzyme bands. The anodal isoenzyme products of the Hdh-1 l o c u s (A*A* and A 2 A 2 ) , and i n t e r m e d i a t e product of the Hdh-2 l o c u s (BB), are l i k e l y supernatant HDH. The ready a s s o c i a t i o n of p e p t i d e s from the two l o c i t o form h y b r i d dimers i s i n d i c a t i v e of the r e l a t i v e l a c k of e v o l u t i o n a r y divergence c h a r a c t e r i s t i c of mu l t i p l e supernatant l o c i ( B a i l e y e t a l . . 1970)., The c o i n c i d e n t e l e c t r o p h o r e t i c m o b i l i t i e s o f a supernatant HDH (such as 30 s t i c k l e b a c k BB i s hypothesized to be) and a i t o c h o n d r i a l forms (such as s t i c k l e b a c k M lX and a 2X may be) has been observed before (Clayton e t a l . , 1973). I n f a c t , the e n t i r e Gasterosteus p a t t e r n as d e s c r i b e d and i n t e r p r e t e d here i s s t r i k i n g l y s i m i l a r to those d e s c r i b e d by C l a y t o n e t a l . . (1973) f o r wal l e y e and sauger. L i k e s t i c k l e b a c k s , t h e s e s p e c i e s possess two supernatant l o c i , one h i g h l y polymorphic and coding f o r the most anodal isozymes ( l i k e s t i c k l e b a c k Mdh-1), and one monomorphic and coding f o r an isozyme o f in t e r m e d i a t e m o b i l i t y ( l i k e Mdh-2). I n walleye, the mi t o c h o n d r i a l enzymes are c a t h o d a l t o a l l supernatant forms (as are the s t i c k l e b a c k MlM* and M 2M 2 bands), while the sauger possessed a m i t o c h o n d r i a l isozyme t h a t c o i n c i d e d 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 with the most cat h o d a l isozyme of supernatant MDH (as do M^X and M 2X). Phosphog1ucoisomerase The multi-banded e l e c t r o p h o r e t i c p a t t e r n of Ga s t e r p s t e u s PGI ( F i g . 3) i s s i m i l a r to t h a t e x h i b i t e d by other bony f i s h e s (Avise and K i t t o 1973). Two s t r u c t u r a l genes code f o r PGI pepti d e s o f d i f f e r i n g e l e c t r o p h o r e t i c m o b i l i t i e s , Pgi-1 f o r the f a s t e r - m i g r a t i n g one and Pgi-2 f o r the slower. PGI i s a dimer, and the bands A 1A* and B*B 1 i n F i g . 3 r e p r e s e n t the two homodimeric isozymes produced by the most common a l l e l e s a t each o f the l o c i (Pgi-1»oo and Pgi-2 »oo r e s p e c t i v e l y ) . ,-Ihe band A » B 1 r e p r e s e n t s the h e t e r o d i m e r i c isozyme o f in t e r m e d i a t e m o b i l i t y . , The l i g h t - s t a i n i n g bands CC and DD anodal t o the B 1 B 1 isozyme band, and EE anodal t o the Aifi* isozyme band, a re 31 F i g u r e 3, The t h r e e s p i n e s t i c k l e b a c k PGI isozyme banding p a t t e r n s . I n d i v i d u a l s homozygous a t the Pgi-1 l o c u s f o r P g i -1*°o (nos. 1, 3 and 5 - 8 ) d i s p l a y the A 1 A* band, those homozygous f o r P g i - I 1 0 5 (no. 2) d i s p l a y the A 2 A 2 band, and Pgi-'1«oo/pgi-1 *05 heterozygotes (no.4) d i s p l a y the A»A», A 1 A 2 and A 2A 2 bands., S t i c k l e b a c k s homozygous a t the Pgi-2 l o c u s f o r P g i - 2 1 0 0 (nos. 1 - 4, 6 and 8) d i s p l a y the B»B*, CC and DD bands, those homozygous f o r P g i - 2 1 * 7 (no. 7) d i s p l a y t h e B 2 B 2 band, and P g i - 2 1 0 0 / P g i - 2 1 4 r h e t e r o z y g o t e s (no. 5) d i s p l a y . t h e B»Bi, B X B 2 and B 2 B 2 bands.. The A^B 1, A 4 B 2 , A 2B* and A Z B 2 bands rep r e s e n t h y b r i d isozymes p o s s e s s i n g one p e p t i d e from each o f the Pgi-1 and Pgi-2 l o c i . 3I3L 1 2 3 4 5 6 7 8 32 • s a t e l l i t e bands' (Avise and K i t t o 1973) . These enzymes are not g e n e t i c a l l y d i s t i n c t from the B i B 1 and A *B 1 isozymes, but i n s t e a d l i k e l y r e s u l t from the o x i d a t i o n of v a r i o u s PGI s u l f h y d r y l groups (Noltmann 1975) . .They a r e c h a r a c t e r i s t i c of the e l e c t r o p h o r e t i c p a t t e r n s of PGI (Avise and K i t t o 1973, Gracy 1975) and other enzymes (Turner et a l . , 1975, Dawson and Green 1975) . The most common v a r i a n t a l l e l e a t the Pgi-2 l o c u s , P g i -2 1 * 7 , codes f o r a p e p t i d e that i n homodimeric form (B 2B 2) c o i n c i d 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 , and hence g e l p o s i t i o n , with the DD band. The h e t e r o d i m e r i c isozyme of a P g i - 2 1 0 0 / P g i -2 1 * 7 heterozygote (B 1 B 2) migrates at the same r a t e as the CC band ( F i g . 3). However, the c h a r a c t e r i s t i c 1:2:1. r a t i o of s t a i n i n g i n t e n s i t y e x h i b i t e d by t h e B 1 B 1 , B 1 B 2, B 2 B 2 bands i n the heterozygote r e a d i l y d i s t i n g u i s h i t from the P g i - 2 1 0 0 homozygote posse s s i n g the B 1 B 1 , CC, DD bands o f i d e n t i c a l m o b i l i t i e s but d i f f e r e n t s t a i n i n g i n t e n s i t y r a t i o s ( i . e . the CC and DD bands s t a i n d i s t i n c t l y l e s s d a r k l y than the B 1 B 1 band). The presence o f d i f f e r e n t a l l e l e s a t the Pgi-2 l o c u s i n P g i - 2 1 0 0 / P g i - 2 1 * 7 heterozygotes r e s u l t s i n the p r o d u c t i o n of a second h y b r i d isozyme A*B 2 which i s anodal to A*B 1. The p o s i t i o n of the klBz isozyme band i s i d e n t i c a l t o the of the EE s a t e l l i t e band of P g i - 2 1 0 0 horaozygotes, but i s c h a r a c t e r i z e d by a f a i n t anodal smear. I n p o p u l a t i o n s i n which the P g i - 2 1 * 7 i s present at s u f f i c i e n t l y high f r e q u e n c i e s , P g i - 2 1 * 7 homozygotes occur, which l a c k both the B 1 B 1 and B 1 B 2 isozyme bands ( F i g . 3) . The absence o f the A 1 B 1 isozyme band i n P g i - 2 1 * 7 homozygotes i s conspicuous. The P g i - 2 1 * 7 a l l e l e i s present i n both L i t t l e Campbell 33 l e i u r u s and trachurus p o p u l a t i o n s a t a low frequency, and r e s u l t s of the l a b cro s s e s support the i n t e r p r e t a t i o n of v a r i a b i l i t y i n the PGI-2 r e g i o n as that due t o two a l l e l e s s e g r e g a t i n g at a s i n g l e autosomal l o c u s (Table 5)., I n most c r o s s e s , both parents were homozygous f o r P g i - 2 1 0 0 , and t h e r e f o r e possessed t h e B l B 1 isozyme, and a l l o f f s p r i n g of thes e c r o s s e s a l s o possessed only the B 1B l isozyme. I n two of the c r o s s e s i n v o l v i n g t r a c h u r u s , and one i n v o l v i n g l e i u r u s , one parent was a P g i - 2 l 0 ° / P g i - 2 1 * 7 heterozygote and d i s p l a y e d a l l t h r e e of the B l B » , B 1 B 2 and B 2 B 2 isozyme bands. One-half of t h e progeny of each o f these c r o s s e s were homozygous f o r the P g i -2ioo a l l e l e and possessed only the B*B* isozyme, w h i l e the other h a l f were heterozygous f o r P g i - 2 1 0 0 / P g i - 2 * * 7 and possessed the B*B», B*B 2 and B 2 B 2 isozymes. The isozyme band A^a 1 r e p r e s e n t i n g the homodimeric a s s o c i a t i o n o f p e p t i d e s coded f o r by the P g i - 1 1 0 0 a l l e l e a t the Pg i - 1 l o c u s was not we l l r e s o l v e d under the e l e c t r o p h o r e t i c c o n d i t i o n s o f t h i s study. The isozyme band was ai d e and tended t o smear towards the anode ( F i g . . 3 ) . T h i s made the v a r i a b i l i t y d i f f i c u l t to score; however, some p o p u l a t i o n s d i d possess a v a r i a n t a l l e l e P g i - 1 l 0 S t h a t coded f o r a peptide wiich i n homodimeric form (A 2A 2) migrated s l i g h t l y f a s t e r than A U 1 . The c l o s e l y spaced A*A l, A l a 2 , A 2 A 2 bands of a P g i -1 1 o o / p g i - 1 1 os heterozygote o f t e n are d i f f i c u l t t o d i s t i n g u i s h from the wide smear of the a 1 a 1 band i n a P g i - 1 1 0 0 homozygote. The A 2 B 1 h y b r i d isozyme band o f heterozygotes i s a l s o d i f f i c u l t t o d e t e c t because i t i s s i m i l a r to the EE band of homozygotes. However, l i k e the AVB 2 isozyme band i t tended t o extend a n o d a l l y 34 Table 5. I n h e r i t a n c e o f Pgi-2 a l l e l e s i n L i t t l e Campbell g i v e r t r a c h u r u s and l e i u r u s s t i c k l e b a c k s . I n both s e t s of cr o s s e s th=> f a s t e r a l l e l e (F) i s P g i - 2 » » 7 and the slower a l l e l e (Sj i s P g i -2»°o. P a r e n t a l genotypes i n brackets were i n f e r r e d from progeny g e n c t y p i c r a t i o s . P a r e n t a l Genotypes No. of Crosses Progeny FF FS SS P a r e n t a l Genotypes No. Of Crosses Progeny FF FS SS Trachurus o* SS FS ? SS 10 0 0 167 ? SS 1 0 6 4 o* (SS) o* SS J tSS) 4 0 0 56 ? FS 1 0 4 6 L e i u r u s o* SS 5 0 0 281 ? SS 35 and c o u l d u s u a l l y be d i s t i n g u i s h e d from the EE band on t h i s b a s i s . N e v e r t h e l e s s , the frequency of the Pgi-1 * ° s a l l e l e , never high to begin with, may be underestimated i n some p o p u l a t i o n s because of s c o r i n g d i f f i c u l t i e s . The a l l e l e was not d e t e c t e d i n e i t h e r of the L i t t l e Campbell p o p u l a t i o n s (parents or progeny) and t h e r e f o r e i t s i n h e r i t a n c e c o u l d not be s t u d i e d . However, the occurrence of p u t a t i v e P g i - I 1 0 5 homozygotes i n a few p o p u l a t i o n s supports the g e n e t i c i n t e r p r e t a t i o n of t h i s v a r i a b i l i t y . These i n d i v i d u a l s possess f a s t - m i g r a t i n g A 2 A 2 and h2B* isozyme bands and, as expected, l a c k the A*A*, A 1A 2 and A*B* bands. Other a l l e l e s occur a t the Pgi-1 l o c u s i n even lower f r e q u e n c i e s . These a l l e l e s produce the c h a r a c t e r i s t i c 3-banded v a r i a b i l i t y i n the PGI-1 region i n heterozygous c o n d i t i o n , and a s i n g l e band of a l t e r e d m o b i l i t y when homozygous. Phosphoglucomutase The e l e c t r o p h o r e t i c p a t t e r n of Gasterosteus PG8 i s the most v a r i a b l e of a l l enzymes examined. F i v e anodal bands with d i s t i n c t e l e c t r o p h o r e t i c m o b i l i t i e s occur i n v a r y i n g f r e q u e n c i e s among p o p u l a t i o n s , and each f i s h possessed one or two of these bands ( F i g . 4). T h i s i s c o n s i s t e n t with the suggestion t h a t PGM i s a monomeric enzyme ( J o s h i e t a l . 1967)- and each band i s the product of a d i f f e r e n t a l l e l e of a s i n g l e autosomal gene. I n d i v i d u a l s d i s p l a y i n g only one band are homozygous f o r one a l l e l e , 2-banded i n d i v i d u a l s a r e heterozygotes p o s s e s s i n g two d i f f e r e n t a l l e l e s . The most common a l l e l e , termed p g m - 1 © 0 , codes f o r the 36 p e p t i d e which s t a i n s as band A (Pig. 4 ) . ,Pgm 1 0 3, the next most common a l l e l e , codes f o r the peptide of band B, which migrates t o a p o s i t i o n s l i g h t l y anodal t o A. The a l l e l e s Pgm 9 3 and Pgta*o code f o r the peptides s t a i n i n g as bands C and D r e s p e c t i v e l y , and Pgm 8 0 f o r the slow-migrating peptide of band £. „ A f t e r prolonged s t a i n i n g or i n c u b a t i o n i n e l e v a t e d c o n c e n t r a t i o n s of glucose-1-phosphate, a second zone of PGM a c t i v i t y (PGM-1) can be detected i n the g e l r e g i o n anodal to t h a t c o n t a i n i n g t h e isozymes monitored i n t h i s study (PGM-2). A l t hough t h i s r e g i o n was c l e a r l y polymorphic, low a c t i v i t y precluded i t s use. , The L i t t l e Campbell t r a c h u r u s p o p u l a t i o n possesses the Pgjiioo a l l e l e (F) and, at a lower frequency, the Pgm9<> a l l e l e (S) (Table 6) . C r o s s e s i n v o l v i n g a l l p o s s i b l e p a r e n t a l genotypic combinations of homo- and heterozygotes confirmed that the isozyme bands observed are indeed under the c o n t r o l of two codominant a l l e l e s a t a s i n g l e autosomal l o c u s . , The L i t t l e Campbell l e i u r u s p o p u l a t i o n c o n t a i n s the Pgm 1 0 3 (F) and the Pgm*o° (S) a l l e l e s (Table 6). Homo- and heterozygotes f o r t h e s e a l l e l e s were employed as par e n t s i n the l a b c r o s s e s , and i n each case t h e o f f s p r i n g d i s p l a y e d genotypic r a t i o s i n accordance with Mendelian p r e d i c t i o n s based on t h e premise t h a t these are two a l l e l e s of a s i n g l e gene. I n h e r i t a n c e p a t t e r n s of the Pgm*3 and Pgm 8 0 a l l e l e s , not present i n the L i t t l e Campbell p o p u l a t i o n s , c o u l d not be examined. However, as with the confirmed a l l e l e s , t h e i r presence i n a homozygous or heterozygous s t a t e r e s u l t s i n the production of e i t h e r one or two isozyme bands. F u r t h e r support f o r the proposed g e n e t i c i n t e r p r e t a t i o n of the observed v a r i a b i l i t y i s 37 F i g u r e 4., The PGM isozyme banding p a t t e r n s o f t h r e e s p i n e s t i c k l e b a c k s . The Pgm10<> a l l e l e codes f o r the p e p t i d e of band k, Pgrn* 0 3 f o r t h a t o f band B, Pgm 9 3 f o r t h a t of band C, Pgm 9« f o r t h a t of band D and Pgm 8 ° f o r t h a t of band E. Homozygotes (nos. 3 and 4) f o r any a l l e l e d i s p l a y the s i n g l e band formed by the p e p t i d e product of t h a t a l l e l e . Heterozygotes (nos. . 1 , 2, 5 and 6) d i s p l a y the two bands formed by the r e s p e c t i v e peptide products of t h e i r two a l l e l e s . 1 2 3 4 5 6 38 Table 6,,, I n h e r i t a n c e o f Pgm a l l e l e s i n L i t t l e Campbell fiiver t r a c h u r u s and l e i u r n s s t i c k l e b a c k s . I n the trac h u r u s c r o s s e s , the f a s t e r a l l e l e (F) i s P g m 1 0 0 and the slower a l l e l e (S) i s P g m 9 » . In the l e i u r u s c r o s s e s , F i s Pgm 1 0 3 and S i s Pgm 1 0 0. Pa r e n t a l genotypes i n bracket s were i n f e r r e d from progeny genotypic r a t i o s . P a r e n t a l Genotypes Progeny P a r e n t a l Genotypes Progeny Hale Female FF FS SS Hale Female FF FS SS Trachurus (1) FF O ) FF 16 0 0 (8) FS (9) SS 0 5 6 (2) FS (2) FS 15 30 17 (8) FS (10) FF 5 5 0 (3) FS (3) FF 4 6 0 (9) SS (9) SS 0 0 6 (4) FF (4) FF 14 0 0 (9) SS (10) FF 0 11 0 (5) (FF) (5) (FF) 16 0 0 (10) FF (11) FF 10 0 0 (5) (FF) (6) (FS) 11 12 0 (10) FF (12) FS 4 3 0 (6) FF (7) FS 4 6 0 (11) FF (12) FS 5 5 0 (7) FS (8) FF 10 10 0 (12) (FS) (13) FF 4 6 0 L e i u r u s 03) FS (15) FS 16 23 17 (15) FF (18) FS 20 19 0 (13) FS (16) FF 29 18 0 (16) FS (19) SS 0 27 31 (14) FF (17) FF 77 0 0 39 t h a t a l l e l e and genotype f r e q u e n c i e s c a l c u l a t e d from the isozyme handing p a t t e r n s i n d i c a t e t h a t p o p u l a t i o n s are i n Hardy-Weinberg e q u i l i b r i u m . Other r a r e a l l e l e s are present i n some p o p u l a t i o n s . The v a r i a b i l i t y observed at the Pgm locus of G a s t e r o s t e u s c l o s e l y resembles, both i n g u a n t i t y and i n nature, t h a t of other v e r t e b r a t e s . PGM i s c h a r a c t e r i s t i c a l l y heterogeneous w i t h i n s p e c i e s , and single-banded homozygous and double-banded heterozygous phenotypes are t y p i c a l (see, f o r example, Roberts e t a l - 1969, Lush 19 69, Otter and Hodgins 1970). L a c t a t e Dehydrogenase Most f i s h s p e c i e s possess three s t r u c t u r a l l o c i coding f o r p o l y p e p t i d e u n i t s of l a c t a t e dehydrogenase that are commonly r e f e r r e d t o as Ldh-A, Ldh-B and Ldh-C (Markert e t a l . 1975) . In t e l e o s t s , e x p r e s s i o n of the A and B l o c i , although v a r i a b l e between s p e c i e s , i s g e n e r a l l y widespread throughout body t i s s u e s , while e x p r e s s i o n o f t h e C l o c u s i s u s u a l l y c o n f i n e d t o n e u r a l t i s s u e such as eye and b r a i n , o r , i n a few s p e c i e s , t h e l i v e r . LDH i s a tetramer and i n v e r t e b r a t e s other than f i s h the p e p t i d e s coded f o r by the A and B genes a s s o c i a t e randomly t o produce b i n o m i a l p r o p o r t i o n s of the f i v e isozymes 4A, 3A1B, 2A2B, 1A3B, 4B. In many f i s h s p e c i e s , however, the a s s o c i a t i o n o f p e p t i d e s coded f o r by the two genes i s r e s t r i c t e d , and not a l l f i v e isozymes are formed. Some s p e c i e s possess o n l y the two homotetrameric isozymes 4A and 4B, others these two homotetramers p l u s the h y b r i d isozyme 2A2B. , Moreover, i n some advanced t e l e o s t s , e x p r e s s i o n of the B l o c u s i s r e s t r i c t e d to a no few t i s s u e s , so t h a t i n many t i s s u e s only the 4A isozyme i s present. T h i s r e d u c t i o n of B gene a c t i v i t y o c c u r s i n some f a m i l i e s of the order Perciformes, and a l l examined f a m i l i e s i n the o r d e r s P l e u r o n e c t i f o r m e s and Tetraodontiformes (Markert et a l . 1975) . The Gasterosteus muscle LDH p a t t e r n c o n s i s t s of a s i n g l e isozyme band. T h i s i s most l i k e l y the 4A isozyme, i n d i c a t i n g t h a t i n Gasterosteus, as i n the forementioned advanced t e l e o s t s , t h e r e i s a r e d u c t i o n i n B gene a c t i v i t y . In t h e present study, t h e s t i c k l e b a c k muscle 4A isozyme d i s p l a y e d an extreme l a c k o f e l e c t r o p h o r e t i c a l l y - d e t e c t a b l e v a r i a b i l i t y . .,0 nly one f i s h , i n the thousands examined, possessed a v a r i a n t a l l e l e o f Ldh-A. The isozyme p a t t e r n o f t h i s i n d i v i d u a l was 5-banded, i n d i c a t i n g t h a t s t i c k l e b a c k LDH-A i s indeed t e t r a m e r i c and t h a t the two A pe p t i d e s produced by the normal and v a r i a n t a l l e l e s a s s o c i a t e d t o produce a l l p o s s i b l e t e t r a m e r i c forms. The l a c k of LDH-A v a r i a b i l i t y was apparent i n the L i t t l e Campbell t r a c h u r u s and l e i u r u s c r o s s e s . , The parents and progeny of a l l c r o s s e s possessed the 4A isozyme band o n l y . When young f i s h were ground up whole, or when a d u l t eye t i s s u e was employed, more LDH isozymes were d e t e c t e d . The eye p a t t e r n c o n s i s t s of f i v e bands, much f a i n t e r than the muscle 4A band. The s l o w e s t - m i g r a t i n g o f these bands i s c a t h o d a l and the f a s t e s t anodal to the 4A band, i n d i c a t i n g t h a t these two bands r e p r e s e n t the 4B and 4C homotetrameric enzymes. The i n t e r m e d i a t e bands r e p r e s e n t the hybr i d B-C isozymes. V a r i a b i l i t y i n t h i s banding p a t t e r n i n d i c a t e d t h at u n l i k e the A gene, one or both of the B and C genes i s polymorphic. Markert e t a l . (1975) i n F i g . 41 4, p. 111, i n d i c a t e t h a t the C gene i s indeed expressed i n the eye, r a t h e r than l i v e r , of f i s h i n the order G a s t e r o s t e i f o r m e s , but do not s t a t e which s p e c i e s were examined., I s o c i t r a t e Dehydrogenase Two forms of NADP-dependent IDH, coded f o r by separate s t r u c t u r a l genes, a r e present i n v e r t e b r a t e t i s s u e (Henderson 1968, Q u i r o z - G u t i e r r e z and Ohno 1970). As with HDH, one type f u n c t i o n s p r i m a r i l y i n the mitochondria, the other i n the c e l l cytoplasm; and d i s t r i b u t i o n of the two types v a r i e s among t i s s u e s . In mammals, m i t o c h o n d r i a l IDH predominates i n h e a r t t i s s u e , supernatant IDH i n l i v e r and kidney (Henderson 1968). S t u d i e s with f i s h r e v e a l c o n s i d e r a b l e v a r i a b i l i t y i n the t i s s u e s p e c i f i c i t y of the two forms among s p e c i e s ( Q u i r o z - G u t i e r r e z and Ohno 1970, Engel e t a l . 1971, H e i n i t z 1977, A l l e n d o r f e t a l . 1975, Shaklee et a l . 1974). However, one p a t t e r n that was common to a l l v e r t e b r a t e s examined i n the above s t u d i e s , with the e x c e p t i o n of the s u r f smelt, was the g r e a t e r anodal m o b i l i t y of supernatant than of m i t o c h o n d r i a l IDH. IDH i s a dimer, and the c h a r a c t e r i s t i c 3-banded phenotype i s d i s p l a y e d by supernatant IDH heterozygotes of mammaliam (Henderson.1968) and most f i s h ( Q u i r o z - G u t i e r r e z and Ohno 1970, Engel e t a l . , 1971) s p e c i e s . , However, R e i n i t z (1977) r e p o r t e d a 2-banded phenotype i n rainbow t r o u t heterozygous f o r supernatant IDH. Gasterosteus muscle samples, run on B u f f e r I I and s t a i n e d f o r ; IDH, d i s p l a y one o f two p a t t e r n s ( F i g . , 5). The f i r s t c o n s i s t s of a s i n g l e , d a r k l y - s t a i n i n g isozyme band. A; the 42 F i g u r e 5. Threespine s t i c k l e b a c k IDH isozyme banding p a t t e r n s . Females (nos. 1 , 3 and 4) d i s p l a y the s i n g l e A band, males (nos. 2 and 5) the A, B and C bands.. 4.23L 1 2 3 4 5 43 Table 7. Sex r a t i o s i n l a b - r e a r e d broods of L i t t l e Campbell R i v e r t r a c h u r u s and l e i u r u s s t i c k l e b a c k s as determined by IDH e l e c t r o p h o r e t i c p a t t e r n s . Cross Progeny C r o s s Progeny Males Females Males Females Trachurus 1 25 18 8 3 7 2 3 7 9 0 6 3 5 5 10 3 8 4 6 10 11 6 4 5 7 3 12 4 3 6 5 15 13 6 4 7 4 7 14 3 7 TOTAL : 80 Males 104 Females L e i u r u s 15 32 27 18 21 21 16 24 20 19 33 25 17 30 47 TOTAL : 140 Males 140 Females 44 second possesses, i n a d d i t i o n to A, a second, more c a t h o d a l band B which migrates h a l f as f a r from the o r i g i n as does A. I n t h i s second phenotype the A and B bands each s t a i n approximately h a l f as d a r k l y as does the s i n g l e A band of the f i r s t phenotype. In a d d i t i o n , the second phenotype possesses a very l i g h t l y s t a i n i n g band, C, which i s l o c a t e d on the o r i g i n . , M u l t i p l e bands t r a i l i n g a n o d a l l y from the A band are o f t e n present, and l i k e l y represent c o n f o r m a t i o n a l isomers or c h e m i c a l l y a l t e r e d forms of the A isozyme. „, No other v a r i b i l i t y was observed i n IDH banding p a t t e r n s among samples assayed i n t h i s s t u d y . , E a r l y i n t h e survey the two p a t t e r n s were r e c o g n i z e d as s e x - s p e c i f i c . Females d i s p l a y the single-banded and males the 3-banded phenotype. Male and female Ga s t e r o s t eu s i n breeding c o n d i t i o n are e a s i l y d i f f e r e n t i a t e d , males possess b r i g h t red t h r o a t s , females are g r a v i d with eggs. Whenever such f i s h were p r e s e n t i n the e l e c t r o p h o r e t i c samples, sex was recorded and checked against IDH phenotype. / Moreover, a f t e r muscle samples had been removed f o r e l e c t r o p h o r e s i s from the non-breeding f i s h of s e v e r a l p o p u l a t i o n s , they were d i s s e c t e d and the presence of o v a r i e s or t e s t e s recorded. I n no case was there a di s c r e p a n c y between the sex of the f i s h as determined m o r p h o l o g i c a l l y and as r e v e a l e d by IDH phenotype. Thus the v a r i a b i l i t y i n IDH isozyme bands r e f l e c t s a sexual dimorphism r a t h e r than a l l e l i c v a r i a t i o n a t an autosomal l o c u s , as i t was i n t e r p r e t e d by Avise (1976) . No f i s h examined i n the present study d i s p l a y e d a v a r i a n t phenotype, i n d i c a t i n g t h a t IDH i s monomorphic i n Ga s t e r o st e u s (although Avise (1976) d i d r e p o r t one sample t h a t a p p a r e n t l y possessed onl y the B band) . n5 The i n h e r i t a n c e s t u d i e s u s i n g L i t t l e Campbell B i v e r f i s h r e v e a l e d t h a t s t i c k l e b a c k s as young as f o u r weeks o l d d i s p l a y both p a t t e r n s . T a b l e 7 presents the sex r a t i o s based on IDH phenotype o f progeny of the c r o s s e s . Although the trachurus c r o s s e s show a n o n s i g n i f i c a n t excess of females (X 2=3.13, P>0.05), the l a r g e r sample s i z e s of the l e i u r u s c r o s s e s r e v e a l a 1:1 r a t i o . Whether t h i s dimorphism i s the r e s u l t of a s e x - s p e c i f i c d i f f e r e n c e at an IDH l o c u s i t s e l f , or a t some other l o c u s a f f e c t i n g the enzyme product of the IDH l o c u s has not been determined. However, s u c c e s s f u l hormonal treatments employed to a l t e r the s e x u a l development of s t i c k l e b a c k s ^ i . e . cause genotypic females to develop i n t o phenotypic males and v i c e verse) a p p a r e n t l y d i d not a f f e c t the banding p a t t e r n s of i n d i v i d u a l s (unpub. , data) . T h i s i n d i c a t e s t h a t the 5 and C bands o f males are not brought about by p o s t - t r a n s l a t i o n a l enzyme m o d i f i c a t i o n s mediated by the male p h y s i o l o g i c a l environment as was t h e case with a s e x - i n f l u e n c e d e l e c t r o p h o r e t i c v a r i a n t of glucose-6-phosphate dehydrogenase i n D r o s o p h i l a (Nomina 1968). S e v e r a l p o s s i b l e e x p l a n a t i o n s remain. The B and C band isozymes may be the products of an autosomal s e x - l i m i t e d gene th a t i s expressed o n l y i n genotypic males (presumably XI i n G a s t e r o s t e u s ) . , The phenotypic f e m i n i z a t i o n (by hormonal treatment) of g e n o t y p i c males does not prevent e x p r e s s i o n of t h i s gene, j u s t as the phenotypic m a s c u l i n i z a t i o n of genotypic females does not induce i t . Such a gene has been d e s c r i b e d i n D r o s o p h i l a by Fukunaga e t a l . (1975) and Tanaka et a l . (1976)., M a l e l e s s (mle) i s a r e c e s s i v e gene l o c a t e d on the autosomal 46 second chromosome. When homozygous, t h i s gene i s l e t h a l i n males but not i n females. The use of sex-transforming genes t o a l t e r the phenotypic sex of both genotypic male and female D r o s o p h i l a d i d not a l t e r the s p e c i f i c i t y o f l e t h a l i t y . I n d i v i d u a l s with one X chromosome, whether p h e n o t y p i c a l l y male or female, d i e d ; while i n d i v i d u a l s with two X chromosomes, whether p h e n o t y p i c a l l y male o r female and with or without an e x t r a ¥ chromosome, s u r v i v e d . In a s i m i l a r f a s h i o n , the B and C isozymes i n Gasterosteus may be the products of an autosomal gene that i s expressed i n genotypic males, even when t h e s e f i s h have undergone hormbnally-induced f e m i n i z a t i o n . A l t e r n a t i v e l y , the gene of the B and C isozymes may be l o c a t e d on a m a l e - s p e c i f i c chromosome, p o s s i b l y t h e f i r s t Y - l i n k e d e l e c t r o p h o r e t i c gene to be d e s c r i b e d f o r any organism. There i s no obvious reason f o r a sexual dimorphism i n Gasterosteus muscle t i s s u e IDH isozymes. Although polymorphisms f o r supernatant IDH have been reported f o r a number of f i s h s p e c i e s , no s e x - l i n k e d or s e x - l i m i t e d e f f e c t s have been noted.... The c e l l u l a r o r i g i n (supernatant or m i t o c h o n d r i a l ) of the s t i c k l e b a c k IDH isozymes of t h i s study was not determined, nor were IDH p a t t e r n s i n t i s s u e s other than muscle examined. A p o s s i b i l i t y worth examining i s t h a t t h e A band c o n s t i t u t e s supernatant IDH, while the c a t h o d a l B and C bands of the male a r e m i t o c h o n d r i a l IDH which has a d i f f e r e n t p a t t e r n of t i s s u e e x p r e s s i o n i n the female. 47 SECTION I I . GENETIC RELATIONS HIPS IN GASTEROSTEUS AC OLE A Ttl S R e s u l t s My a n a l y s i s o f g e n e t i c v a r i a b i l i t y i n 79 n a t u r a l p o p u l a t i o n s of G. a c n l e a t a s i s presented i n two p a r t s . Since an e v a l u a t i o n of the s i g n i f i c a n c e o f d i f f e r e n c e s i n a l l e l e f r e q u e n c i e s and d i s t r i b u t i o n s among, p o p u l a t i o n s i s p o s s i b l e only by comparison with the amount of i n t r a p o p u l a t i o n v a r i a b i l i t y , the a n a l y s i s 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 among p o p u l a t i o n s i s preceded by an examination of polymorphism, h e t e r o z y g o s i t y and a l l e l e d i s t r i b u t u i o n s w i t h i n p o p u l a t i o n s . G e n e t i c V a r i a b i l i t y w i t h i n P o p u l a t i o n s For a l l p o p u l a t i o n s , the sample s i z e , a l l e l e f r e q u e n c i e s , observed and expected numbers of heterozygotes, and l e v e l of h e t e r o z y g o s i t y f o r each of the s i x polymorphic l o c i a re presented i n Appendices I I to V I I . Tab l e 8 c o n t a i n s a summary of the i n t r a p o p u l a t i o n v a r i a b i l i t y . Polymorphism B i t h i n each p o p u l a t i o n , a l o c u s was designated polymorphic i f the frequency o f t h e most common a l l e l e was l e s s than, or eq u a l t o , 0.99. LDH and IDH were monomorphic i n a l l p o p u l a t i o n s . LDH possessed a s i n g l e e l e c t r o p h o r e t i c a l l y d i s t i n g u i s h a b l e a l l e l e and IDH d i s p l a y e d an i n v a r i a n t but s e x u a l l y dimorphic 4 8 Table 8. Summary of i n t r a p o p u l a t i o n g e n e t i c v a r i a b i l i t y i n Gasterosteus a c u l e a t u s . . P o p u l a t i o n Average No. Polymorphic H e t e r o z y g o t e s 1 Average Genes Sampled L o c i Obs (Exp) H e t e r o z y g o s i t y (± S.E.) (± S.E.) Marine ENGLS 59.8 ± 0. 7 4 17 (17) 0. 082 ± 0. 043 HOESB2 6 2. 0 ± 23. 9 2 12 (11) 0. 044 ± 0. 026 MITLG 63.5 ± 12. 4 5 23 (21) 0. 088 ± 0. 047 LC 19 95.3 ± 2. 0 4 58 (53) 0. 117 ± 0. 065 COHIB 54.0 ± 0. 0 3 18 (16) 0. 073 ± 0. 051 HDERA2 41.3 ± 19. 0 4 20 (19) 0. 107 0. 0 50 SOOKP 64.8 1. 4 5 24 (23) 0. 093 ± 0. 037 KOKSB 134.5 ± 3. 5 4 53 (55) 0. 101 0. 045 SLMNC 106.0 ± 0. 0 5 48 (58) 0. 147 ±' 0. 066 GRAPI 82.0 ± 0. 0 6 38 (41) 0. 13 0 ± 0. 051 BAMFS 67.8 ± 0. 7 6 40 (4 4) 0. 170 0. 0 47 SAME 87.8 ± 0. 7 5 47 (4 9) 0. .139 ± 0. 0 44 HAINL 46.0 ± 0. 0 4 19 (22) 0. 122 ± 0. 052 CONGE 78.3 ± 7. 2 6 40 (38) 0. 127 ± 0. 0 31 SANMA 88.0 ± 12. 3 5 46 (46) 0. 124 -± 0* 044 CHEHR 32.0 ± 0. 0 5 18 (18) o. 137 ± 0. P51 Large Lake COHIL 74.0 ± 0, 0 4 19 (22) 0. 082 ± 0. 050 SARIL 114.0 ± 0. 0 5 88 (97). 0. 213 ± 0 . 068 SARIN 22.0 ± 0. 0 5 14 (10) 0. 127 ± 0. 055 SPEOT 275.5 ± 0. 8 4 167 (178) 0. 162 ± 0. 0 75 MCBT1 509.8 ± 34. 0 4 300 (319) 0. 153 m 0. 069 GBCEN 105.5 ± 1. 3 5 60 (69) 0. 202 ± 0. 077 STELL 37.0 ± 1. 0 5 22 (21) 0. 148 ± 0. 0 53 BOBTL 4 2.0 ± 0. 0 2 13 (11) 0. 068 ± 0. 058 Small Lake CHE ML 16 2.8 ± 3. 5 3 53 (51) 0. 07 9 0. 043 FULLL 70.3 ± 4. 1 4 73 (67) 0. 24 7 ± 0. 089 MABNL 149.8 ± 0. 7 3 59 (57) 0. 09 5 ± 0. Q 48 LKERR 92.0 ± 0. 0 4 47 (42) 0. 115 ± 0. 060 HOTEL 228.0 ± 0. 0 5 123 (128) 0. 142 ± 0. 076 KLEIN 90.0 t 0. 0 4 53 (4 9) 0. 140 ± 0. 082 PAQLK 82.0 ± 0. 0 2 19 (21) 0. 065 ± 0. 054 TBO UT 100.0 ± 0. 0 4 63 (57) 0. 144 ± 0. 066 GABBY 28.0 ± 0. 0 2 13 (10) 0. 088 ± 0. 0 73 BLACK 509.8 ± 3. 9 3 265 I [288) 0. 144 ± 0. 084 PATER 213.5 ± 0. 9 2 80 177) 0. 092 ± 0. 075 DEVIL 169.3 ± 1. 0 1 46 (42) , 0. 062 ± 0. 058 SUMNL 131.3 ± 1. 4 1 43 (46) 0. 087 ± 0. 0 77 MODLK 118.0 ± 1. 3 1 23 (2 3) 0. 050 ± 0. 046 LOWBL 94.0 ± 0. 0 3 22 (24) 0. 058 ± 0. 048 MCOYL 259.5 ± 0. 9 4 41 (43) 0. 04 1 ± 0. 0 18 CECIL 205.8 ± 0. 7 3 118(118) 0. 143 ± 0. 067 HO R GL 161.3 ± 1. 4 2 36 (34) 0. 053 ± 0, 040 49 OBMBD 124.0 ± 0.0 2 21 (20) 0.041 .,+ 0.029 FAB WL 277.5 ± 0.9 4 182(176) 0.159 -± 0.Q85 CEDAR 216.0 ± 1.0 2 105 (10 0) 0. 118 "±; 0.074 PAXTB 220.3 t 13. 0 5 76 (75) 0.086 •± 0.047 PAXTL 87.3 ± 2.0 6 67 (68) 0.201 i ' 0.066 ENOSB 230.5 ± 41. 1 3 63 (64) 0.09 6 ,± 0.0 59 EHOSL 307.0 t 2.0 4 143(149) 0.121 0.061 GOOSE 131.8 ± 2.7 4 79 (7 8) 0. 151 0.067 HECHL 70.3 6. 3 4 46 (49) 0.186 ± 0.085 CBAHL 95.3 ± 1.0 2 51 (47) 0.126 i 0.077 L o u - l y i n g Stream or • Swamp SK00S 3 61.0 ± 23. 1 3 9 (10) 0.Q42 0.025 SHIPC 54.0 ± 0.0 4 16 (19) 0.088 ± 0.0 42 LC 18 173.8 ± 0.7 4 113 (112) 0.161 0.072 GIFFS* 22.8 ± 8.6 3 6 (6) 0.061 • ± 0.025 TEXAS 49.8 ± 4.5 5 31 (29) 0. 157 ,± 0.063 PATBS 98.0 ± 0.0 4 79 (86) 0.221 0.091 EBEYI 136.0 t 0.0 4 91 (10 1) 0. 188 ± 0.075 SKAGI 74.0 ± 0.0 6 44 (47) 0.19 0 ± 0.076 MOOSE 71.5 ± 1. 3 6 50 (49) 0.175 ± 0.068 SMODL 148.3 •± 20.5 3 111 {112) 0. 180 ± 0.082 NATHNS 76.0 ± 26. 6 4 61 (60) 0.251 • ± 0.098 CLQTZ 94.0 ± 0.0 4 67 (65) 0.173 ± 0.066 HCBPD 199.0 ± 1. 4 5 134 (128) 0. 161 ± 0.066 SABIJ 76. 3 + 3.7 4 43 (54) 0.184 ± 0.072 TYNEP 625.2 ± 27.3 4 340 (373) 0.15 1 0.068 HOQOB 66.0 ± 0.0 4 19 (20) 0.075 ± 0.038 OTTER 233.3 ± 1.4 3 86 (87) 0. 106 ± 0.0 56 FARBL 343.8 ± 0.7 4 207 (211) 0. 154 ± 0.081 I s o l a t e d Stream o r Swamp KEOGH 90.0 ± 0.0 1 27 (29) 0.031 0.0 76 FABSW 149.8 0.7 3 64 (71) 0.122 ± 0.063 SPBPD 145.8 ± 0.7 3 71 (78) 0.059 ± 0.046 SLZEE 103.0 ± 32.9 3 6 4 (74) 0. 152 ± 0.071 DBYBN 100.0 ± 0.0 1 23 (25) 0.062 ± 0,058 Mixed FBASB 208.0 ± 6.6 4 103 (108) 0.132 ± 0.068 LABDC 227.5 ± 32.7 5 165(168) 0.194 ± 0.069 FOLCE 126.0 ± 0.0 4 58 (6 0) 0.133 0.0 58 LC 40 38.8 ± 14.7 5 43 (49) 0.144 ± 0.068 1 Excludes data f o r Pgi-1 l o c u s . 2 M i s s i n g data f o r Ck. , 3 H i s s i n g data f o r Mdh-3. * Missing data f o r Pgm. 5 M i s s i n g data f o r P g i - 2 . 50 banding p a t t e r n . Of the polymorphic l o c i , a l l but Pgm were se g r e g a t i n g f o r two major a l l e l e s . , Pgm possessed f i v e common a l l e l e s . Hare a l l e l e s were detected a t a l l polymorphic l o c i except Mdh-3. The number of polymorphic l o c i per p o p u l a t i o n ranged from 1 to 6 (12.5% to 75% of the 8 examined) . The average l e v e l of polymorphism, c a l c u l a t e d as the unweighted average over a l l p o p u l a t i o n s ( e x c l u d i n g mixed samples, samples from l a k e s c o n t a i n i n g two p a r t i a l l y or c ompletely d i s t i n c t p o p u l a t i o n s , and samples f o r which data f o r one or more l o c i were m i s s i n g ) , was 3.76 (47%) (Table 8) . H e t e r o z y g o s i t y For a s i n g l e g e n e t i c l o c u s , h e t e r o z y g o s i t y i s g i v e n by: n H = 1 - Z ] p , 2 , where n i s t h e number of a l l e l e s a t the l o c u s , i = i and p, i s the frequency of the i*k- a l l e l e i n the p o p u l a t i o n . H e t e r o z y g o s i t y p r o v i d e s a measure of i n t r a p o p u l a t i o n v a r i a b i l i t y t h a t accounts not only f o r the a b s o l u t e number of a l l e l e s of a gene (which i s polymorphism), but a l s o t h e i r frequency i n the p o p u l a t i o n . . Q u i t e simply, i t i s the p r o p o r t i o n of i n d i v i d u a l s heterozygous at a l o c u s , assuming t h a t the p o p u l a t i o n i s i n Hardy-Weinberg e q u i l i b r i u m . , In t h i s study, l e v e l s of h e t e r o z y g o s i t y v a r i e d among l o c i and among p o p u l a t i o n s . Appendices I I t o VII give the l e v e l of h e t e r o z y g o s i t y f o r each polymorphic l o c u s i n each p o p u l a t i o n . Idh and Ldh were c o n s i s t e n t l y monomorphic, so the value of h e t e r o z y g o s i t y f o r these l o c i was 0 f o r a l l p o p u l a t i o n s . F o r polymorphic l o c i possessing two major a l l e l e s the maximum value 51 of h e t e r o z y g o s i t y i s 0.5. , For Pgm, with f i v e a l l e l e s , the maximum value i s 0.8. The presence of rare a l l e l e s a t any l o c u s i n c r e a s e s the p o t e n t i a l l e v e l o f h e t e r o z y g o s i t y f o r t h a t l o c u s . H e t e r o z y g o s i t y f o r Ck ranged from 0.000 to 0.497 with an average value of 0.107 i n the 78 p o p u l a t i o n s examined (Appendix VI) . For Pgi-1 the values ranged from 0.000 t o 0.294 i n 76 p o p u l a t i o n s , with an average of 0.018. T h i s value i s undoubtedly low because of undetected Pgi-1 heterozygotes i n many p o p u l a t i o n s (Sect. I ) . F o r Pgi-2, the l i m i t i n g values were 0.000 and 0.526, with an average value of 0.Q85 f o r 78 p o p u l a t i o n s . H e t e r o z y g o s i t y at the Mdh-1 l o c u s v a r i e d between 0.000 and 0.500, averaging 0.101 over 79 p o p u l a t i o n s . The range f o r Mdh-3 was from 0,000 to 0.500, with an average f o r 77 p o p u l a t i o n s of 0.275.. For Pgm, h e t e r o z y g o s i t y ranged from 0.Q00 t o 0.665, with an average of 0,418 over 78 p o p u l a t i o n s . , For each p o p u l a t i o n , the average h e t e r o z y g o s i t y (H) i s the average of the i n d i v i d u a l h e t e r o z y g o s i t y values f o r a l l l o c i examined. The l e v e l of average h e t e r o z y g o s i t y ranged from 0.041 i n McCoy (HCOYL) and Ormond (QBMND) lakes to 0.247 i n F u l l e r Lake (FULLL) (Table 8) , The unweighted average value o f H over a l l p o p u l a t i o n s ( e x c l u d i n g mixed samples, samples from l a k e s c o n t a i n i n g two p a r t i a l l y or completely d i s t i n c t p o p u l a t i o n s , and samples f o r which data f o r one or more l o c i were missing) was 0. 124. Hardy-weinberg E q u i l i b r i u m For each polymorphic l o c u s i n each p o p u l a t i o n the observed 52 number o f het e r o z y g o t e s and the number expected under Hardy-Weinberg e q u i l i b r i u m c o n d i t i o n s are giv e n i n Appendices I I t o V I I . The G - t e s t , employing l o g l i k e l i h o o d r a t i o s (Sokal and Rohlf 1969), was used t o t e s t t he s i g n i f i c a n c e of the departure o f observed genotypic f r e q u e n c i e s from Hardy-Weinberg e x p e c t a t i o n s f o r a l l l o c i (except Pgi-1) s u f f i c i e n t l y polymorphic to generate t h r e e g e n o t y p i c c l a s s e s each c o n t a i n i n g at l e a s t 5 i n d i v i d u a l s w i t h i n a p o p u l a t i o n . In the case of Pgm, genotypic c l a s s e s with expected v a l u e s of l e s s than 5 were lumped (homozygotes with homozygotes, heterozygotes with h e t e r o z y g o t e s ) . Because a l l e l e f r e q u e n c i e s tended t o remain con s t a n t i n p o p u l a t i o n s sampled more than once (see below), data f o r these p o p u l a t i o n s were lumped i n order t o enable a s i n g l e t e s t f o r each l o c a t i o n (Appendices I I t o V I I ) . With the e x c e p t i o n of repeated samples obtained from the S e r p e n t i n e R i v e r (TYHEP) which are not i n c l u d e d i n these r e s u l t s but discussed s e p a r a t e l y below, independent t e s t s on m u l t i p l e samples from a s i n g l e l o c a t i o n d i d not g i v e d i f f e r e n t r e s u l t s . Genotypic r a t i o s f o r Pgi-1 were not t e s t e d a g a i n s t Hardy-Beinberg p r e d i c t i o n s because of the low frequency o f a l l e l e s other than t h e common P g i - I 1 0 0 at t h i s l o c u s , and the d i f f i c u l t y of d e t e c t i n g them i n heterozygous form (Sect. . I) . Omissions i n the s c o r i n g of heterozygotes i s the most l i k e l y e x p l a n a t i o n f o r the observed heterozygote d e f i c i e n c y f o r PGI-1 i n many p o p u l a t i o n s (Appendix IV). For c r e a t i n e kinase, an o v e r a l l G - t e s t on e i g h t polymorphic p o p u l a t i o n s i n d i c a t e d no departure of observed genotypic f r e q u e n c i e s from Hardy-Weinberg e q u i l i b r i u m ( X 2 = 3 . 7 4 , d . f . = 8 , 53 P>0.90). When p o p u l a t i o n s sere t e s t e d independently, each with one degree of freedom, none d e v i a t e d at the 5% l e v e l from p r e d i c t e d genotypic p r o p o r t i o n s . An o v e r a l l G -test on seven p o p u l a t i o n s polymorphic f o r Mdh-1 again i n d i c a t e d c l o s e agreement of observed with expected r a t i o s (X 2=3.77, d.f.=7, P>0.80) . Independent t e s t s on each of the p o p u l a t i o n s r e v e a l e d no s i g n i f i c a n t departures from expected v a l u e s . , For the h i g h l y polymorphic Mdh-3 l o c u s , the o v e r a l l t e s t on 32 p o p u l a t i o n s was s i g n i f i c a n t (X 2=50.7, d. f. = 32, P<0.05), and when te s t e d independently t h e genotypic r a t i o s of three p o p u l a t i o n s d e v i a t e d from Hardy-Beinberg e x p e c t a t i o n s at the 5% l e v e l (Appendix I I ) . , The t h r e e samples rep r e s e n t widely d i f f e r i n g h a b i t a t s and geographic l o c a t i o n s . One (SARIL) was c o l l e c t e d from S a r i t a Lake, a l a r g e l a k e on the west coast of Vancouver I s a l n d , one (EBEYI) from a l o w - l y i n g slough near the mouth of the Snohomish R i v e r i n northwestern Washington and one (FARSW) from an i s o l a t e d swamp t o the north of, and d r a i n i n g i n t o , F a r e w e l l Lake on eas t e r n Vancouver I s l a n d . Three out of f i f t y i s a s l i g h t l y g r e a t e r p r o p o r t i o n than the 5% o f samples expected t o d e v i a t e from p r e d i c t e d values by chance a l o n e , and a l l three p o p u l a t i o n s show heterozygote d e f i c i e n c i e s . T h i s c o n d i t i o n suggests the Wahlund e f f e c t (a shortage of heterozygotes r e s u l t i n g from the i n c l u s i o n of i n d i v i d u a l s from more than one p o p u l a t i o n with d i f f e r i n g gene f r e q u e n c i e s i n a s i n g l e sample).. However, i t c o u l d a l s o r e s u l t from nonrandom breeding among d i f f e r e n t Mdh-3 genotypes w i t h i n a s i n g l e p o p u l a t i o n , or from d i s r u p t i v e , or v a r i a b l e , s e l e c t i v e f o r c e s a c t i n g upon a s i n g l e p o p u l a t i o n . 54 Four p o p u l a t i o n s polymorphic f o r Pgi-2 possessed genotypic r a t i o s i n f u l l accordance with Hardy-Weinberg p r e d i c t i o n s (X2-3.35, d.f.=4, P>0.50). When tested independently none o f - t h e p o p u l a t i o n s d e v i a t e d from expected v a l u e s . For 37 p o p u l a t i o n s polymorphic f o r the m u l t i - a l l e l i c Pgm l o c u s , the o v e r a l l G - t e s t showed no departure of observed from expected g e n o t y p i c r a t i o s (X2=70.Q7, d . f . f 6 4 , P>0.30). Two p o p u l a t i o n s i n the Bear R i v e r system on Vancouver I s l a n d , one (8CRTL) from a l a r g e l a k e and one (FARWL) from a s m a l l l a k e , possessed g e n o t y p i c f r e q u e n c i e s s i g n i f i c a n t l y d i f f e r e n t from Hardy-Weinberg c o n d i t i o n s when t e s t e d independently (Appendix V I I ) . T h i s number i s no more than expected from sampling e r r o r . However, each of these l o c a t i o n s was sampled twice and i n n e i t h e r case d i d the a l l e l i c or genotypic r a t i o s vary between samples. The flcCreight Lake samples possessed f i v e a l l e l e s , and the d e v i a t i o n from e q u i l i b r i u m c o n d i t i o n s r e s u l t e d from a d e f i c i e n c y of heterozygotes p o s s e s s i n g the two most common a l l e l e s . The F a r e w e l l Lake samples possessed f o u r a l l e l e s , and t h e r e was a d e f i c i e n c y i n two of the heterozygous c l a s s e s and a s u r p l u s i n two of the ot h e r s . M u l t i p l e Samples Samples from the p o p u l a t i o n s examined i n t h i s study were c o l l e c t e d by a v a r i e t y of methods, and a t d i f f e r e n t times of the year. Gene frequency v a r i a b i l i t y among p o p u l a t i o n s may simply r e f l e c t d i f f e r i n g gear s e l e c t ! v i t i e s , or seasonal f l u c t u a t i o n s i n gene f r e q u e n c i e s . S e v e r a l Vancouver I s l a n d l a k e p o p u l a t i o n s 55 were sampled s i m u l t a n e o u s l y by minnow t r a p s and pole s e i n e (or d i p net) to determine i f d i f f e r e n t gear types d i s p r o p o r t i o n a t e l y sample d i f f e r e n t p o p u l a t i o n segments which possess d i s t i n c t gene f r e q u e n c i e s . In g e n e r a l , minnow t r a p s c a t c h only t h e l a r g e r members of a s t i c k l e b a c k p o p u l a t i o n , whereas p o l e seines e f f e c t i v e l y sample the s m a l l e r s i z e c l a s s e s bat not the f a s t e r -swimming and o f f s h o r e - d w e l l i n g l a r g e r f i s h . F i s h caught by the two methods i n Blackwater Lake (BLACK) were subsampled, and t h e i r h y p u r a l l e n g t h recorded before e l e c t r o p h o r e s i s . The mean; length of trap-caught s t i c k l e b a c k s was 5.07 cm (N=43, S.E.^?1.4) and of p o l e - s e i n e d s t i c k l e b a c k s was 3.46 cm (N=40, S.E. =1.2). For each p o p u l a t i o n sampled by the two methods, a G value was obtained f o r the d i f f e r e n c e i n a l l e l e frequency between the two samples f o r each polymorphic enzyme. The sum of the G values obtained f o r each enzyme was t e s t e d f o r goodness of f i t to the X 2 d i s t r i b u t i o n (Table 9) . D i f f e r e n c e s i n sex r a t i o s (as determined by the IDH banding patterns) between trapped and s e i n e d samples were a l s o t e s t e d . As Table 9 i n d i c a t e s , Mdh-1, Pgi-2 and Ck were each polymorphic i n one p o p u l a t i o n , Mdh-3 was polymorphic i n t h r e e , and Pgm i n a l l four o f the p o p u l a t i o n s sampled. There were no d i f f e r e n c e s a t the 5% s i g n i f i c a n c e l e v e l i n a l l e l e f r e q u e n c i e s between trap-caught and p o l e - s e i n e d samples f o r any of these enzymes. Fr e q u e n c i e s of the s e x u a l l y dimorphic banding p a t t e r n i n d i c a t e d t h a t sex r a t i o s were a l s o s i m i l a r among f i s h sampled by the two methods. Thus, t h e r e i s no evidence t h a t the f i s h caught by d i f f e r e n t methods c o n s t i t u t e g e n e t i c a l l y d i s t i n c t subgroups of a p o p u l a t i o n . However, i f such v a r i a b i l i t y does e x i s t w i t h i n p o p u l a t i o n s , 56 d i f f e r e n c e s i n a l l e l e f r e q u e n c i e s between age groups might be expected. Although s t i c k l e b a c k s are d i f f i c u l t to age with c e r t a i n t y , an attempt was made to t e s t t h i s h y p othesis. S t i c k l e b a c k s c o l l e c t e d by pole seine from a roadside slough one mile e a s t of O t t e r Point i n Sooke, Vancouver I s l a n d (OTTER) comprised two d i s t i n c t s i z e c l a s s e s , small (mean length=3.74 cm, N=66, S.E.=0.04) and l a r g e (mean leagth=4.?2 cm, N=51, S.E.=0.10). The pronounced b i m o d a l i t y of the s i z e d i s t r i b u t i o n i n t h i s p o p u l a t i o n suggests the presence of two d i s t i n c t age c l a s s e s , with the l a r g e r f i s h c o n s t i t u t i n g the o l d e r age group. Three enzymes were polymorphic i n the f i s h from t h i s l o c a t i o n , HDH-3, PGM and CK, but t h e r e were no d i f f e r e n c e s i n a l l e l e f r e q u e n c i e s between the l a r g e and s m a l l f i s h (Table 9) . Thus, to t h e e x t e n t t h a t d i s t i n c t s i z e c l a s s e s are i n d i c a t i v e of d i s t i n c t age c l a s s e s , no d i f f e r e n c e s i n gene f r e q u e n c i e s between age groups i s i n d i c a t e d . I f such d i f f e r e n c e s do e x i s t i n -. th e OTTER p o p u l a t i o n ; they are too s m a l l f o r present sample s i z e s to r e v e a l . To d e t e c t s e a s o n a l v a r i a t i o n i n a l l e l e f r e q u e n c i e s w i t h i n p o p u l a t i o n s , s e v e r a l Vancouver I s l a n d lake p o p u l a t i o n s i n the Bear E i v e r and Somass R i v e r watersheds were sampled i n both s p r i n g (May or June) and f a l l (Septempber) 1978. F o r a s i n g l e l o c a t i o n , m u l t i p l e samples taken s i m u l t a n e o u s l y by d i f f e r e n t methods i n e i t h e r the s p r i n g or the f a l l were combined f o r t h i s comparison (previous r e s u l t s i n d i c a t e d no a l l e l e frequency d i f f e r e n c e s between f i s h sampled by the v a r i o u s methods). The Hay and June samples were mainly a d u l t f i s h caught i n s h o r e i n breeding c o n d i t i o n . Small, newly-hatched f i s h (young-of-year) 57 Table 9,. A l l e l e frequency v a r i a b i l i t y i n m u l t i p l e samples from Gast e r o s t e u s p o p u l a t i o n s . G values were c a l c u l a t e d f o r a l l e l e d i s t r i b u t i o n s at i n d i v i d u a l l o c i among m u l t i p l e samples. The c r i t i c a l c h l - s q u a r e v a l u e at t h e .05 l e v e l f o r each comparison i s given i n b r a c k e t s . Mdh-1 Mdh-3 Pgi-2 Pgm Ck Ida A. Vancouver I s l a n d p o p u l a t i o n s : p o l e - s e i n e vs. minnow t r a p No. p o p u l a t i o n s : 1 3 1 4 1 4 Degrees freedom: 1 3 1 11 1 4 Obs. G value: 2.62 2.97 1.68 9.44 0.41 4.97 C r i t . Chi-sguare: (3.84) (7.81) (3.84) (19.7) (3.84) (9.49) B. Slough eas t of O t t e r P o i n t : l a r g e vs. s m a l l f i s h Degrees freedom: — 1 — 2 1 1 Obs. G value: — 2.18 — 2.68 0. 19 0.43 C r i t . Chi-square: — (3.84) — (5.99) (3.84) (3.8 4) C. .Vancouver I s l a n d p o p u l a t i o n s : May/June vs. September capture No. p o p u l a t i o n s : 2 9 5 12 ~ 14 Degrees freedom: 2 9 5 26 —• 14 Obs. G v a l u e : 2.46 9.26 2. 10 32.0 — 47. 5*** C r i t . Chi-square: (5.99) (16.9) (11. 1) (38.9) — (23.7) D. Serpentine B i v e r pond: samples over time Degrees freedom: — 4 4 8 4 4 Obs. G value: — 40.3*** 3.01 3.76 5.85 3.10 C r i t . Chi-square: — (9.49) (9.49) (15.5) (9.49) (9.49) 58 E. Chemainas and Marion l a k e s : m u l t i p l e samples Degrees freedom: 3 3 3 — 3 Obs. G v a l u e : 4.95 — 1.32 0.74 ^- 3.60 C r i t . Chi-square: (7.81) — (7.81) (7.81) — (7.81) F. Enos Lake: 1977 vs. 1978 Benthic samples Degrees freedom: — — 1 1 1 Obs. G value: — — 1.88 — 0.16 . 0.70 C r i t . Chi-sguare: — — (3.84) — (3.84) (3.84) L i m n e t i c samples Degrees freedom — 1 1 2 1 1 . Obs. G v a l u e : — 6.26* 0.80 6.95* 0.07 91.2*** C r i t . C h i -sguare: — (3.84) (3.84) (5.99) (3.84) (3.84) * P < 0.05 ** P < 0.001 *** P < 0.0001 5 9 c o n s t i t u t e d a s i g n i f i c a n t p o r t i o n of the September samples, although a d u l t f i s h were s t i l l present., Again, f o r each polymorphicsenzyrae i n each p o p u l a t i o n sampled, a G value was obtained f o r the d i f f e r e n c e i n a l l e l e f r e q u e n c i e s between the s p r i n g and f a l l samples. .The sum of the G values obtained f o r each enzyme was t e s t e d f o r goodness of f i t to the X 2 d i s t r i b u t i o n * MDH-1 was polymorphic i n two p o p u l a t i o n s , PGI - 2 i n f i v e , MDH-3 i n n i n e and PGM i n twelve p o p u l a t i o n s . None of these enzymes show a s i g n i f i c a n t d i f f e r e n c e (at the 5 % l e v e l ) i n a l l e l e f r e g u e n c i e s between t h e s p r i n g and f a l l samples (Table 9 ) . A h i g h l y s i g n i f i c a n t d i f f e r e n c e i n the f r e q u e n c i e s of the two IDH banding p a t t e r n s (P<0.001) i n d i c a t e s that t h e sex r a t i o s were not constant between sampling p e r i o d s . , When examined i n d i v i d u a l l y , each with one degree of freedom, 3 o f the 14 p o p u l a t i o n s r e v e a l s i g n i f i c a n t d i f f e r e n c e s i n sex r a t i o between s p r i n g and f a l l . One p o p u l a t i o n had a l a r g e r p r o p o r t i o n of females i n June, two c o n t a i n e d a g r e a t e r number of females i n t h e f a l l . However, as s t a t e d e a r l i e r and f u r t h e r supported by t h i s example, a l l e l e f r e q u e n c i e s at the polymorphic l o c i examined i n t h i s study do not d i f f e r between the sexes, so t h a t v a r i a b l e sex r a t i o s are not i n d i c a t i v e of v a r y i n g a l l e l e f r e q u e n c i e s (except, perhaps, a t any sex-determining l o c i t h a t may e x i s t ) . To f u r t h e r examine the p o s s i b i l i t y of a l l e l e freguency v a r i a b i l i t y over time, a s i n g l e p o p u l a t i o n i n the F r a s e r V a l l e y (TYNEP) was sampled s e v e r a l times over a 15 month p e r i o d . The l o c a t i o n c o n s i s t s of a s m a l l , h i g h l y e u t r o p h i c pond adjacent to the headwaters of t h e Serpentine B i v e r . The pond i s f l o o d e d by 60 the r i v e r i n the winter and i s o l a t e d d u r i n g the d r i e r summer months. In the summer i t s h r i n k s to a f r a c t i o n of i t s winter s i z e . T h i s pond possesses a dense s t i c k l e b a c k p o p u l a t i o n which must f l u c t u a t e i n numbers with v a r i a t i o n s i n pond s i z e , and which probably experiences both immigration and emi g r a t i o n d u r i n g f l o o d p e r i o d s . ,.Thus t h e pond seemed a l i k e l y l o c a t i o n to observe changes i n a l l e l e f r e q u e n c i e s over time, e i t h e r i n response t o v a r i a b l e s e l e c t i v e regimes, or t o random events such as g e n e t i c d r i f t o r the d i f f e r e n t i a l immigration of genotypes. Samples were p o l e s e i n e d from the pond i n March 1978 ( a d u l t s ) , June 1978 (separate c o l l e c t i o n s o f a d u l t s and young-of-year) , august 1978 (combined c o l l e c t i o n of a d u l t s and young) and June 1979 ( a d u l t s ) . There was an average of 44 f i s h per sample. Pgi-1 and Hdh-1 were monomorphic f o r the same a l l e l e i n a l l samples. P g i - 2 , Mdh-3, Ck and Pgm were polymorphic, and f o r each of these l o c i a G value was obtained f o r the a l l e l e frequency d i s t r i b u t i o n s i n the f i v e samples (Table 9). Only t h e value o b t a i n e d f o r Mdh-3 was s i g n i f i c a n t (P<0.001), i n d i c a t i n g t h a t a l l e l e f r e q u e n c i e s at t h e other three l o c i remained constant over time. At the Mdh-3 l o c u s , the Mdh-S 1 0 0 a l l e l e was present a t r e l a t i v e l y high f r e q u e n c i e s i n the March 1978 and June 1978 (young-of-year) samples (0.26 and 0.23) , and at much lower f r e q u e n c i e s i n the June 1978 ( a d u l t ) , August 1978 and June 1979 samples (0.09, 0.06 and 0.06) . T h i s a p p a r e n t l y r e f l e c t s a r e a l change i n a l l e l e f r e q u e n c i e s oyer time, and although the cause cannot be a s c e r t a i n e d i t w i l l be shown i n l a t e r s e c t i o n s t h a t Mdh-3 a l l e l e s may be s u b j e c t t o n a t u r a l s e l e c t i o n i n Gasterosteus p o p u l a t i o n s . , The n o n s i g n i f i c a n t r e s u l t f o r IDH 61 (Table 9) i n d i c a t e s t h a t t h e sex r a t i o d i d not vary among samples. Further evidence f o r the s t a b i l i t y of gene f r e q u e n c i e s over s h o r t time p e r i o d s i s provided by samples from Chemainus Lake (CHEML) on Vancouver I s l a n d and Marion (Jacobs) Lake (HARNL) i n the 0. B. C. ^Research F o r e s t near Haney, B.C. Marion Lake, at an e l e v a t i o n of 1000 f t above sea l e v e l , contained no s t i c k l e b a c k s p r i o r to an i n t r o d u c t i o n i n 1974 of 4000 Chemainus Lake Gasterosteus (J.D. McPhail, pers. comm.).. Chemainus Lake was sampled with minnow t r a p s i n 1977 and 1978, and Marion Lake i n 1977 and 1979. Mdh-3, Pgi-1 and Ck were monomorphic f o r the same a l l e l e i n a l l f o u r samples. Mdh-1, Pgi-2 and Pgm were polymorphic. G values c a l c u l a t e d on the a l l e l e f r e q u e n c i e s f o r each o f these enzymes i n the f o u r samples are not s i g n i f i c a n t a t the 5% l e v e l (Table 9) . T h i s i n d i c a t e s t h a t not o n l y was t h e r e no change i n a l l e l e f r e g u e n c i e s w i t h i n e i h e r of the l a k e s over a 1 or 2 year p e r i o d , but t h a t no d i f f e r e n c e s i n a l l e l e f r e g u e n c i e s developed between the l a k e s , even a f t e r the two p o p u l a t i o n s had been separated f o r s e v e r a l years. , Thus, over t h i s s h o r t time span t h e r e i s no evidence f o r the a c t i o n of g e n e t i c d r i f t o r n a t u r a l s e l e c t i o n upon the g e n e t i c v a r i a b i l i t y d e tected by e l e c t r o p h o r e t i c methods. To t h i s p o i n t , the s t i c k l e b a c k s i n h a b i t i n g a s i n g l e lake have been c o n s i d e r e d a s i n g l e p o p u l a t i o n , and the samples taken from a s m a l l p o r t i o n o f a l a k e r e p r e s e n t a t i v e of i t s e n t i r i t y . The data presented so f a r support t h i s i d e a t o the extent that m u l t i p l e samples taken over t i m e , and i n Cedar Lake (CEDAR) on Vancouver I s l a n d over c o l l e c t i n g s i t e s s e v e r a l hundred yards 6 2 a p a r t , s e r e homogeneous. However, i n t r a - l a k e p o p u l a t i o n s u b d i v i s i o n remains a t h e o r e t i c a l p o s s i b i l i t y , e s p e c i a l l y i n l a r g e Vancouver I s l a n d l a k e s with t h e i r wide ranges o f h a b i t a t d i v e r s i t y ( i . e . .Cowichan, Great C e n t r a l and Sproat l a k e s ) . The d i f f i c u l t i e s i n o b t a i n i n g a c c e s s t o , and i n sampling the p r i m a r i l y o f f s h o r e - d w e l l i n g p o p u l a t i o n s of these l a r g e l a k e s hindered attempts t o d i s c e r n i n t r a - l a k e v a r i a b i l i t y . The r e s t r i c t e d samples obtained from each lake are t h e r e f o r e , by n e c e s s i t y , c o n s i d e r e d r e p r e s e n t a t i v e of the e n t i r e l a k e p o p u l a t i o n (s) . 4 G e n e t i c a l l y d i s t i n c t p o p u l a t i o n s of G., a c u l e a t u s were, however, found to c o e x i s t i n two small l a k e s , Paxtoa l a k e on Texada I s l a n d and Enos Lake on Vancouver I s l a n d . Both l a k e s c o n t a i n two m o r p h o l o g i c a l l y d i s t i n c t groups of s t i c k l e b a c k s , r e f e r r e d t o as b e n t h i c and l i m n e t i c . In Paxton Lake, Larsen (1976) found t h a t the two types d i f f e r e d i n d i s t r i b u t i o n , f e e d i n g behavior and a g g r e s s i v e n e s s , as w e l l as morphology (the number o f body p l a t e s , body s i z e and shape, the number o f g i l l r a k e r s and presence or absence o f p e l v i c g i r d l e ) . The morphology and d i s t r i b u t i o n of the two types i n Enos Lake i s s i m i l a r to those of Paxton (J.p. McPhail, pers. comm.). In t h i s study, the l a r g e r b e n t h i c s t i c k l e b a c k s (PAXTB and EDO SB) were caught by minnow t r a p s i n both l a k e s , and the s m a l l e r l i m n e t i c s t i c k l e b a c k s (P&XTL and ENOSL) by n i g h t - t i m e s e i n i n g . Paxton Lake was sampled i n 1977, Enos i n both 1977 and 1978. , S t a b i l i t y of the a l l e l e f r e q u e n c i e s c h a r a c t e r i z i n g b e n t h i c and l i m n e t i c p o p u l a t i o n s was examined by comparing the 1977 and 1978 samples of Enos Lake., F o r the b e n t h i c f i s h , a l l e l e 63 f r e q u e n c i e s at the polymorphic Pgi-2 and Ck l o c i remained constant from year t o year. For the l i m n e t i c s , , a l l e l e f r e g u e n c i e s were constant between years at the Pgi-2 and Ck l o c i , but d i f f e r e d a t the Mdh-3 (P<0.025) and Pgm (P<0.05) l o c i (Table 9) . The high l e v e l s of average h e t e r o z y g o s i t y d i s p l a y e d by these p o p u l a t i o n s (Table 8), u n c h a r a c t e r i s t i c of p o p u l a t i o n s i n h a b i t i n g s m a l l l a k e s , seems a s s o c i a t e d with the presence of two d i s t i n c t t y p es of Gasterosteus i n c l o s e c o e x i s t e n c e . . Hiechhold (HECHL) and Cranby (CS&NL) l a k e s on Texada I s l a n d , and Goose Lake (GOOSE) i n t h e O.B.C. Research F o r e s t possess s t i c k l e b a c k s d i s p l a y i n g both b e n t h i c - l i k e and l i m n e t i c -l i k e phenotypes, as w e l l as a d i s t r i b u t i o n of phenotypes apparently i n t e r m e d i a t e to the two types (J.D. McPhail, p e r s . comm.). The absence o f c l e a r l y bimodal d i s t r i b u t i o n s of phenotypes i n d i c a t e s that these p o p u l a t i o n s may not be composed o f two completely d i s t i n c t g e n e t i c groups, and samples from these l a k e s are presented as samples from s i n g l e p o p u l a t i o n s . However, there i s l i k e l y a degree of a s s o r t a t i v e mating beween l i k e phenotypes i n these l a k e s as w e l l ; and they a l s o are c h a r a c t e r i z e d by high l e v e l s of average h e t e r o z y g o s i t y (Table 8) . Genetic V a r i a b i l i t y among P o p u l a t i o n s In c o n t r a s t to the g e n e r a l homogeneity of a l l e l e f r e q u e n c i e s w i t h i n p o p u l a t i o n s of G. a c u l e a t u s , comparisons among p o p u l a t i o n s r e v e a l great g e n e t i c h e t e r o g e n e i t y . ,G values c a l c u l a t e d f o r the a l l e l e d i s t r i b u t i o n s a t each of t h e polymorphic l o c i i n a l l 79 p o p u l a t i o n s are given i n Table 10. 64 a l l a r e s i g n i f i c a n t a t the 5% l e v e l , i n d i c a t i n g t h a t t h e r e e x i s t r e a l d i f f e r e n c e s i n a l l e l e f r e q u e n c i e s among p o p u l a t i o n s . I examined p a t t e r n s i n the a l l e l e d i s t r i b u t i o n s , and i n the amount of g e n e t i c v a r i a b i l i t y c h a r a c t e r i z i n g d i f f e r e n t types of p o p u l a t i o n s . Comparison Of Freshwater And Marine P o p u l a t i o n s To determine i f the d i f f e r e n c e s i n l i f e h i s t o r y between marine and freshwater s t i c k l e b a c k s are r e f l e c t e d i n d i f f e r i n g gene f r e g u e n c i e s , a G-test was used t o compare the a l l e l e f r e q u e n c i e s a t each polymorphic l o c u s i n marine and freshwater p o p u l a t i o n s . P o p u l a t i o n s r e s i d i n g permanently i n b r a c k i s h or t i d a l l y - i n f l u e n c e d water were excluded, but marine p o p u l a t i o n s sampled while breeding i n b r a c k i s h e s t u a r i n e r e g i o n s were i n c l u d e d . A t o t a l of 65 p o p u l a t i o n s , 16 marine and 49 freshwater, were examined. A l l e l e f r e g u e n c i e s a t two l o c i d i f f e r e d between the two, Pgm (P<0.005) and Mdh-3 (P<0.001). At t h e Ck, Mdh-1 and Pgi-2 l o c i , there were no d i f f e r e n c e s i n a l l e l e f r e q u e n c i e s between marine and freshwater p o p u l a t i o n s {Table 10). Nei's (1972, 1975) measure o f g e n e t i c d i s t a n c e (D) makes p o s s i b l e an e v a l u a t i o n o f the d i f f e r e n c e s between freshwater and marine p o p u l a t i o n s based on the i n f o r m a t i o n provided by a l l e l e f r e q u e n c i e s at a l l l o c i . .The minimum ge n e t i c d i s t a n c e between two p o p u l a t i o n s , X and Y, i s c a l c u l a t e d as the mean of D = { X j - y , ) 2 / 2 over a l l l o c i , where x, i s the frequency of t h e a l l e l e i n X and y, i s the frequency of the i ^ 1 a l l e l e i n 65 Table 10. A l l e l e frequency v a r i a b i l i t y among G. aculgatus p o p u l a t i o n s . .Values o f average f r e q u e n c i e s are not weighted by sample s i z e . C r i t i c a l c h i - s q u a r e and F values a t .05 l e v e l are i n b r a c k e t s . Average A l l e l e F r e q u e n c i e s Populations Mdh-1 (100) Hdh-3 (100) Pgi-2 (100) Ck (85) Pgm (100) Pgm (90) Pgm (103) Marine 0.953 0. 147 0.941 0.955 0.759 0.183 0.046 A l l Freshwater 0.923 0.635 0.944 0.879 0.554 0.173 0.21 1 Large Lakes 0.870 0.552 0.945 0.965 0.725 0. 155 0. 10 7 Small Lakes 0.970 0. 653 0.934 0.891 0.501 , 0.183 0.24 2 Low-lying Streams 0.897 0.550 0.959 0.780 0.603 0.155 0. 20 1 I s o l a t e d Streams 0.860 0.563 1.000 1.000 0.674 0.19 1 0.087 O v e r a l l C h i-square: 1423. 1 (99.6) *** 5310. 8 (97.4) *** 867. 8 (97.4) *** 4058.7 (98.5) *** 8947.5 (349. 6) *** Marine-Fresh-water F Value: 0. 44 (3.99) 12.61 (4.00) ** 0.46 (4.00) 1.32 (4.00) 3.95 (2.41) # Among Habitat F Value: 2. 43 (2.52) 4.81 (2.52) * 1.76 (2.52) 9^69 (2.52) *** 2. 14 (2.52) * P < 0.05 ** P < 0.001 *** P < 0.0001 66 Y. Genetic d i s t a n c e s between a l l p a i r s of 16 marine, a l l p a i r s of 41 freshwater and a l l p a i r s of one each of these marine and freshwater p o p u l a t i o n s were c a l c u l a t e d . P o p u l a t i o n s from l a k e s c o n t a i n i n g two completely or p a r t i a l l y d i s t i n c t p o p u l a t i o n s (Paxton, Enos, Goose, Hie.chh.old and Cranby) were excluded from t h i s a n a l y s i s , as were any p o p u l a t i o n s l a c k i n g e l e c t r o p h o r e t i c data f o r one or more l o c i . The average g e n e t i c d i s t a n c e between marine p o p u l a t i o n s was 0.0055 ± 0.0011 (range from 0.0005 t o 0.0239, 120 pairwise comparisons), between freshwater p o p u l a t i o n s was 0.0632 ± 0.0072 (range from 0.0001 t o 0.2345, 820 comparisons) and between marine and freshwater p o p u l a t i o n s was 0.0608 ± 0.0126 (range from 0.0010 to 0.2202, 656 c o m p a r i s o n s ) T h e low g e n e t i c d i s t a n c e between marine p o p u l a t i o n s i n d i c a t e s a great s i m i l a r i t y i n a l l e l e d i s t r i b u t i o n s among them, while the l a r g e r d i s t a n c e between freshwater p o p u l a t i o n s i n d i c a t e s a l l e l i c d i f f e r e n t i a t i o n . The average g e n e t i c d i s t a n c e between marine and freshwater p o p u l a t i o n s was s i m i l a r t o t h a t between p a i r s of freshwater p o p u l a t i o n s , i n d i c a t i n g t h a t , on average, t h e g e n e t i c d i f f e r e n c e s between marine and freshwater s t i c k l e b a c k p o p u l a t i o n s were no g r e a t e r than between two freshwater p o p u l a t i o n s . However, the average marine-freshwater genetic d i s t a n c e was much g r e a t e r than that between p a i r s of marine p o p u l a t i o n s . .. Geographic V a r i a b i l i t y In A l l e l e Freguencies To t e s t f o r c l i n a l v a r i a t i o n i n a l l e l e f r e g u e n c i e s , simple 67 r e g r e s s i o n s of f r e q u e n c i e s of the most common a l l e l e s at polymorphic l o c i were performed, employing l o n g i t u d e and l a t i t u d e as the independent v a r i a b l e s . Frequencies of Mdh-1 1 0 0, Hdh-3 1 0 0 , Ckss, p g i - 2 1 0 0 , Pgm 1 0 0, Pgm 1 0 3 and Pgm*° were s u b j e c t e d to a r c s i n square root t r a n s f o r m a t i o n b e f o r e use. Since a t some l o c i s i g n i f i c a n t a l l e l e frequency d i f f e r e n c e s e x i s t between s t i c k l e b a c k s i n marine and freshwater h a b i t a t s , separate analyses were performed on marine and freshwater p o p u l a t i o n s . Hixed samples and samples from lakes c o n t a i n i n g two p o p u l a t i o n s were excluded. A t o t a l of 16 marine and 51 .. freshwater p o p u l a t i o n s were employed. Among freshwater p o p u l a t i o n s , f r e q u e n c i e s of the Pgm 9 0 (P=0.05, R2=0.07, N=50) and Ck«s (p=0.03, B 2=0.08, N=51) a l l e l e s were s i g n i f i c a n t l y c o r r e l a t e d with l a t i t u d e . ,. The frequency of P g m 9 ° i n c r e a s e d and C k 8 S decreased i n a n o r t h e r l y d i r e c t i o n . Freshwater f r e q u e n c i e s of the remaining a l l e l e s were not c o r r e l a t e d with l a t i t u d e . S i m i l a r l y , only the frequency of C k 8 S (P=0.05, R 2=0.07, N=51) and Pgm 9 0 (P=0.Q 1, R«=0. 12, 8=50) were c o r r e l a t e d with l o n g i t u d e i n freshwater. T h e i r f r e q u e n c i e s i n c r e a s e d and decreased, r e s p e c t i v e l y , i n a w e s t e r l y d i r e c t i o n . None of these c o r r e l a t i o n s sere h i g h l y s i g n i f i c a n t and do not provide strong evidence f o r c l i n a l v a r i a t i o n i n freshwater a l l e l e f r e g u e n c i e s . Only two out of 14 r e g r e s s i o n s had p r o b a b i l i t i e s of l e s s than 0.05, a p r o p o r t i o n which may be a t t r i b u t a b l e to chance alone. In the next s e c t i o n , the freguency o f C k 8 5 i s shown to be s i g n i f i c a n t l y h i g h e r i n l o w - l y i n g streams than i n other freshwater h a b i t a t s . The l a t i t u d i n a l c l i n e i n C k 8 5 frequency probably r e f l e c t s the f a c t t h a t a l l but one of the 68 southern ( i . e . Washington) p o p u l a t i o n s t h a t were sampled came from l o w - l y i n g streams. If samples from Washington l a k e s were i n c l u d e d , t h a t apparent north-south c l i n e i n C k 8 5 frequency would probably disappear. ,On t h e other hand, i t i s p o s s i b l e that the n o r t h e r l y and e a s t e r l y i n c r e a s e i n Pgm 9 0 frequency r e s u l t s from s e l e c t i o n by an environmental v a r i a b l e t h a t d i s p l a y s a s i m i l a r c l i n e , but very l i t t l e of the a l l e l e frequency v a r i a b i l i t y {7% and 12%) i s e x p l a i n e d by these geographic t r e n d s . Among marine p o p u l a t i o n s , none of the a l l e l e s examined were s i g n i f i c a n t l y c o r r e l a t e d with l a t i t u d e . Since the study i n c l u d e d o n l y one marine sample from Washington, the l a t i t u d i n a l range was o b v i o u s l y l i m i t e d and perhaps i n s u f f i c i e n t to allow d e t e c t i o n of c l i n a l v a r i a t i o n . However, f r e q u e n c i e s of Pgm 1 0 3 (P=0.0002, R2=0.64, N=16), Ck 8* (P=0.Q003, R*=0.66, N=15) and Pgi-2»oo (p=0.06, RZ=0.23, N=16) were c o r r e l a t e d with l o n g i t u d e . F r e g u e n c i e s of C k 8 5 and P g i - 2 1 0 0 (both common a l l e l e s ) decreased i n a w e s t e r l y d i r e c t i o n , while the frequency of Pgm 1 0 3 (a l e s s common a l l e l e ) i n c r e a s e d . A l l of the marine samples i n the study except t h e one from Washington S t a t e (CHEHW) were c o l l e c t e d e i t h e r from the west coast of Vancouver I s l a n d (SLMNC, GRAPI, BAMFS, SARIE, HAIN1, CONGR, SANHA) or from the more e a s t e r l y S t r a i t of Georgia, s e p a r a t i n g Vancouver I s l a n d from the B.C. .mainland (ENGLR, HOHSB, SITLG, LC 19, COWIB, HDERA, SOOKP, KOKSH) . , To determine i f the s i g n i f i c a n t r e g r e s s i o n s o f Pgm 1 0 3, C k 8 S and P g i - 2 1 0 0 f r e q u e n c i e s on l o n g i t u d e were the r e s u l t of genetic d i f f e r e n t i a t i o n between these two r e g i o n s , the G-t e s t was used 69 to compare a l l e l e f r e q u e n c i e s at a l l polymorphic l o c i between the pooled samples of each r e g i o n . As expected, s i g n i f i c a n t a l l e l e frequency d i f f e r e n c e s were demonstrated f o r the Pgm (G-56..08, P<0.0001), Pgi-2 (G=11.40, P<0.00 1) and Ck (G=58.48, P=0,00G1) l o c i . No d i f f e r e n c e i n the a l l e l e f r e q u e n c i e s of the two r e g i o n s was apparent at t h e Mdh-1 (G=0.97, P>0.30) or Mdh-3 (G=1.67, P>0.15) l o c i . Comparisons Of A l l e l e F requencies Among H a b i t a t s The r e l a t i v e g e n e t i c homogeneity found among marine p o p u l a t i o n s that share a somewhat uniform o c e a n i c h a b i t a t c o n t r a s t s s h a r p l y with the g e n e t i c h e t e r o g e n e i t y found among po p u l a t i o n s occupying d i v e r s e freshwater h a b i t a t s . T h i s c o n t r a s t suggests the p o s s i b i l i t y of gene-environment a s s o c i a t i o n s . To examine t h i s p o s s i b i l i t y the p o p u l a t i o n s sampled were c l a s s i f i e d i n t o f i v e c a t e g o r i e s a c c o r d i n g t o h a b i t a t type; marine; l a r g e lake (>1 km 2); s m a l l l a k e (<1 km2) ; l o w - l y i n g stream or swamp; and i s o l a t e d stream or swamp {as i n Table 9). Low-lying streams and swamps are those at e l e v a t i o n s l e s s than 100 m above sea l e v e l , and those a t e l e v a t i o n s g r e a t e r than 100 m i n drainage systems with l a k e s or streams known t o c o n t a i n s t i c k l e b a c k s at higher e l e v a t i ons. „ I s o l a t e d streams and swamps a r e those a t e l e v a t i o n s g r e a t e r than 100 m with no d i r e c t or i n d i r e c t inflow from bodies of water known t o c o n t a i n s t i c k l e b a c k s . , , T h i s a p p a r e n t l y s i m p l i s t i c c l a s s i f i c a t i o n of h a b i t a t s a c t u a l l y encompasses a l a r g e number of environmental v a r i a b l e s i n c l u d i n g water fl o w , temperature regimes, v e g e t a t i o n types and number and 70 type of p r e d a t o r s . A l l samples o b v i o u s l y composed of s t i c k l e b a c k s from more than one p o p u l a t i o n (Mixed),. i n c l u d i n g samples from the s m a l l l a k e s p o s s e s s i n g b e n t h i c and l i m n e t i c f i s h , were excluded from the a n a l y s i s . For each of the polymorphic enzymes a one-way a n a l y s i s of v a r i a n c e was performed on the frequency of the most common a l l e l e ( a r c s i n square r o o t transformed) i n each p o p u l a t i o n . , The r e s u l t s are presented i n Tab l e 10. The frequency o f the C k a s a l l e l e was h i g h l y heterogeneous among types of p o p u l a t i o n s (P<0.00001). A Duncan's m u l t i p l e range t e s t ( S t e e l and T o r r i e 1960) i n d i c a t e d t h a t the frequency of C k 8 s was s i g n i f i c a n t l y lower i n l o w - l y i n g stream or swamp p o p u l a t i o n s than i n a l l other types of p o p u l a t i o n s . , The ANQVA r e s u l t s f o r the Mdh-1 a l l e l e approached s i g n i f i c a n c e (P=0.06) . The frequency of t h i s a l l e l e was lower i n p o p u l a t i o n s from l a r g e l a k e s and low- l y i n g streams than from the ocean, small l a k e s and i s o l a t e d streams. The P g i -2ioo a l l e l e was not s i g n i f i c a n t l y heterogeneous (P=0. 15) among h a b i t a t s . ,• The Mdh-3 1 0 0 a l l e l e frequency was heterogeneous (P=0,002)., As shown p r e v i o u s l y , a Duncan's range t e s t i n d i c a t e d t h a t i t s frequency was lower i n marine than i n a l l freshwater p o p u l a t i o n s ; t h e r e was no s i g n i f i c a n t h e t e r o g e n e i t y among the d i f f e r e n t t y p es o f freshwater h a b i t a t s . The ANOTA f o r the frequency of Pgm 1 0 0 approached s i g n i f i c a n c e (P=0«09); the frequency of t h i s a l l e l e was lowest i n small l a k e p o p u l a t i o n s and h i g h e s t i n marine p o p u l a t i o n s . Comparisons Of Ge n e t i c V a r i a b i l i t y Amonq H a b i t a t s 71 The f r e q u e n c i e s of the most common a l l e l e s at a number of l o c i were lowest i n p o p u l a t i o n s i n h a b i t i n g l a r g e l a k e s and low-l y i n g streams and swamps. T h i s i n d i c a t e s t h a t e i t h e r t h e number o r the freguency, or both, o f l e s s common a l l e l e s i n these p o p u l a t i o n s i s h i g h e r than i n those occupying s m a l l l a k e s and i s o l a t e d streams or swamps. To examine t h i s p o s s i b i l i t y an a n a l y s i s of va r i a n c e using the same h a b i t a t c l a s s i f i c a t i o n scheme was performed on the number of polymorphic l o c i per p o p u l a t i o n . P o p u l a t i o n s with data missing f o r one or more l o c i were excluded. The h i g h l y s i g n i f i c a n t r e s u l t (?<0.00001) con f i r m s the presence of h e t e r o g e n e i t y i n the amount of polymorphism among d i f f e r e n t t y p e s of p o p u l a t i o n s . A Duncan's m u l t i p l e range t e s t showed t h a t the number of polymorphic l o c i was s i g n i f i c a n t l y l o v e r i n p o p u l a t i o n s i n h a b i t i n g s m a l l l a k e s and i s o l a t e d streams than i n other p o p u l a t i o n s . , Marine p o p u l a t i o n s possessed the hi g h e s t average number of polymorphic l o c i (4.8) , f o l l o w e d by l a r g e l a k e s and l o w - l y i n g streams (4.3), and f i n a l l y by s m a l l lakes (2.8) amd i s o l a t e d streams (2.2). An a n a l y s i s of variance performed on the l e v e l s o f average h e t e r o z y g o s i t y (values a r c s i n square r o o t transformed) i n p o p u l a t i o n s occupying the f i v e h a b i t a t c a t e g o r i e s was a l s o s i g n i f i c a n t (P=0,001).,, Average h e t e r o z y g o s i t y was high e r i n l a r g e l a k e s and l o w - l y i n g streams than i n other p o p u l a t i o n s . The average value o f H was 0.158 i n l o w - l y i n g streams and swamps, 0.144 i n l a r g e l a k e s , 0.118 i n marine p o p u l a t i o n s , 0.103 i n s m a l l l a k e s and 0.095 i n i s o l a t e d streams and swamps. These r e s u l t s i n d i c a t e that p o p u l a t i o n s i n h a b i t i n g s m a l l l a k e s or i s o l a t e d streams possessed l i t t l e g e n e t i c v a r i a b i l i t y ; 7 2 they were c h a r a c t e r i z e d both by low polymorphism and low h e t e r o z y g o s i t y . Marine p o p u l a t i o n s , the most polymorphic of a l l examined, possessed the g r e a t e s t numbers of v a r i a n t a l l e l e s . , However, they d i s p l a y e d an i n t e r m e d i a t e l e v e l of h e t e r o z y g o s i t y i n d i c a t i n g t h a t these v a r i a n t a l l e l e s d i d not occur a t high f r e g u e n c i e s . The s t i c k l e b a c k s o f l a r g e l a k e and l o w - l y i n g stream p o p u l a t i o n s d i s p l a y e d fewer a l l e l e s than marine p o p u l a t i o n s ( i . e . they had lower polymorphism) but the v a r i a n t a l l e l e s they d i d possess were present at higher f r e g u e n c i e s ( i . e . they had highe r h e t e r o z y g o s i t y ) . Comparisons W i t h i n And Between Drainage Systems Extending the study of p a t t e r n s of g e n e t i c v a r i a b i l i t y , I questioned i f the a l l e l e f r e g u e n c i e s i n d i f f e r e n t types of h a b i t a t s w i t h i n a drainage system were more s i m i l a r to each other than were a l l e l e f r e q u e n c i e s i n the same type of h a b i t a t i n d i f f e r e n t drainage systems. , Two watersheds were sampled e x t e n s i v e l y f o r t h i s purpose, the Somass B i v e r system d r a i n i n g eastward and the Bear B i v e r system d r a i n i n g westward on Vancouver I s l a n d . E i g h t l o c a t i o n s i n the Somass and eleven i n the Bear, r e p r e s e n t i n g a v a r i e t y o f h a b i t a t s , were sampled, many o f them i n both the s p r i n g and f a l l of 1978. Habitat c l a s s i f i c a t i o n s used i n the a n a l y s i s were l a r g e l a k e (>1 km 2), s m a l l la k e (<1 km2) and stream or swamp. A completely nested a n a l y s i s o f v a r i a n c e was performed on the a l l e l e d i s t r i b u t i o n s a t each of the polymorphic l o c i , Pgi-2, Pgm, Mdh-1 and Mdh-3. The l e v e l s of the a n a l y s i s were drainage system, h a b i t a t type w i t h i n drainage system, i n d i v i d u a l l o c a t i o n w i t h i n h a b i t a t type and, f i n a l l y , m u l t i p l e samples from w i t h i n a s i n g l e l o c a t i o n . The r e s u l t s of the a n a l y s i s are given i n Table 11. For every enzyme, the h e t e r o g e n e i t y i n a l l e l e d i s t r i b u t i o n s between l o c a t i o n s was h i g h l y s i g n i f i c a n t (P<0.001) . a l l e l e f r e q u e n c i e s d i d not d i f f e r s i g n i f i c a n t l y between r i v e r systems and v a r i e d between h a b i t a t types only i n the case of Mdh-1 (P<0.05). T h i s a n a l y s i s i n d i c a t e s t h a t the l a r g e amount of g e n e t i c v a r i a b i l i t y observed among freshwater s t i c k l e b a c k p o p u l a t i o n s i s not due to gene frequency d i f f e r e n c e s between drainage systems nor between type of h a b i t a t occupied. N e i t h e r of these f a c t o r s accounted f o r t h e g r e a t v a r i a b i l i t y among l o c a t i o n s . However, the previous a n a l y s i s i n v o l v i n g many more p o p u l a t i o n s showed t h a t the a l l e l e f r e q u e n c i e s of some l o c i were r e l a t e d t o h a b i t a t type, and the n e g a t i v e r e s u l t s of the present a n a l y s i s may be at l e a s t p a r t l y due t o s m a l l sample sizes,,. Examination o f the components of v a r i a n c e (Appendix VIII) i n d i c a t e s that f o r Mdh-3 3H% and f o r Pgm'3 30$ of the v a r i a n c e i n gene f r e q u e n c i e s was due to d i f f e r e n c e s between the two drainaqe systems. The s i g n i f i c a n c e o f these apparent d i f f e r e n c e s c o u l d be t e s t e d by e x t e n d i n g the a n a l y s i s t o i n c l u d e a greater number of watersheds. To examine d i f f e r e n c e s i n a l l e l e f r e q u e n c i e s w i t h i n and between drainage systems using the i n f o r m a t i o n : from a l l l o c i combined, the g e n e t i c d i s t a n c e s between a l l pairwise combinations of p o p u l a t i o n s w i t h i n each of the Bear and Somass systems, and a l l p a i r w i s e combinations of p o p u l a t i o n s between the two systems, were c a l c u l a t e d . The average g e n e t i c d i s t a n c e between p o p u l a t i o n s w i t h i n both the Bear (N=55) and Somass 74 f a b l e 11. a l l e l e frequency v a r i a b i l i t y w i t h i n and between the Bear and Somass watersheds. A completely nested a n a l y s i s of variance was performed on the a r c s i n transformed f r e q u e n c i e s of the most common a l l e l e { s ) at each polymorphic l o c u s . Locus Source of V a r i a b i l i t y Sum of Squares Degrees Freedom Mean Square F R a t i o Proba-b i l i t y Hdh-1 Between systems 0.008 1 . 0. 008 0.08 0.85 among h a b i t a t s 0.290 4 0.072 4.0 0 0.03 among l o c a t i o n s 0.246 13 0.019 9.5 1 0.00 R e s i d u a l 0.024 11 0.002 Mdh-3 Between systems 1.307 1 1. 307 3.99 0.21 among h a b i t a t s 1.096 4 0. 274 1.50 0.25 Among l o c a t i o n s 2.452 13 0. 189 63.05 0.00 Residual 0.033 11 0.003 Pgi-2 Between systems 0.002 1 0. 002 0.08 0.80 Am on g h a h i t at s 0.081 4 . 0.020 1.54 0.25 Among l o c a t i o n s 0.171 13 0.013 6.53 0,00 Residu a l 0.021 11 0.002 Pgm* Between systems 0.341 1 0.341 2.32 0.4 3 Among h a b i t a t s 0.642 4 0. 160 0.83 0.53 Among l o c a t i o n s 2.490 13 0. 192 27.43 0.00 R e s i d u a l 0.076 11 0. 007 Pgm 9 3 Between systems 0.594 1 0.594 : 4.87 0. 19 Among h a b i t a t s 0.490 4 0. 122 0.98 0.50 Among l o c a t i o n s 1,66 9 13 0. 123 42.67 0.00 R e s i d u a l 0.033 11 0.003 75 (N=28) drainages was 0.038. T h i s d i s t a n c e i s lower than the average d i s t a n c e of 0.047 (N=88) f o r comparisons i n v o l v i n g one p o p u l a t i o n from each drainage. I t i s a l s o lower than the p r e v i o u s l y c a l c u l a t e d average freshwater g e n e t i c d i s t a n c e , based on p a i r w i s e comparisons of a l l freshwater p o p u l a t i o n s examined, of 0.063 (N=820). Thus, s t i c k l e b a c k s w i t h i n watersheds do seem t o be e l e c t r o p h o r e t i c a l l y more s i m i l a r to each o t h e r than to other freshwater s t i c k l e b a c k s . The r e l a t i o n s h i p of g e n e t i c d i s t a n c e t o a c t u a l geographic d i s t a n c e between p o p u l a t i o n s w i t h i n a watershed was examined using the e l e v e n l o c a t i o n s sampled i n the Bear R i v e r drainage. Geographic d i s t a n c e between sampling s i t e s was measured along e x i s t i n g waterways., A r e g r e s s i o n a n a l y s i s of a l l pairwise combinations between p o p u l a t i o n s of g e n e t i c d i s t a n c e on geographic d i s t a n c e was h i g h l y s i g n i f i c a n t (R 2 = 0.34, N=55, P<0.0001). T h i s i n d i c a t e s t h a t p o p u l a t i o n s near one another i n t h e drainage system possessed a l l e l e d i s t r i b u t i o n s more s i m i l a r than d i d d i s t a n t p o p u l a t i o n s . , T h i s g e n e t i c s i m i l a r i t y between adjacent l o c a t i o n s suggests the occurrence of gene f l o w , past or p r e s e n t , between p o p u l a t i o n s . I t i s of i n t e r e s t t o d i s c o v e r , then* i f i s o l a t e d p o p u l a t i o n s are l e s s g e n e t i c a l l y v a r i a b l e than other p o p u l a t i o n s due t o r e s t r i c t e d gene exchange. To t e s t t h i s s uggestion the e i g h t l a k e s of the Bear R i v e r system (stream and swamp p o p u l a t i o n s excluded) were d i v i d e d i n t o two groups. The f i r s t c o n s i s t e d of f i v e l a k e s with 0 or 1 l a k e c o n t a i n i n g s t i c k l e b a c k s f l o w i n g i n t o them, the second c o n s i s t e d of three l a k e s with more than 1 s t i c k l e b a c k - c o n t a i n i n g l a k e s f l o w i n g i n t o them, e i t h e r 76 d i r e c t l y or i n d i r e c t l y ( i . e . through another lake) . A t t e s t on the mean l e v e l of average h e t e r o z y g o s i t y (values a r c s i n square r o o t transformed) was s i g n i f i c a n t (t=2.72, d.f.=7, P<0,05). I s o l a t e d l a k e s had lower l e v e l s of average h e t e r o z y g o s i t y than l a k e s with g r e a t e r p o s s i b i l i t i e s of gene i n f l o w . These i s o l a t e d l a k e s i n the upper r e g i o n s o f t h e watershed tend t o be small l a k e s . I n an e a r l i e r s e c t i o n , s m a l l l a k e s were found t o have low l e v e l s of average h e t e r o z y g o s i t y . Thus, i s o l a t e d l a k e s may be l e s s heterozygous because they tend t o be s m a l l , or s m a l l l a k e s may be l e s s heterozygous because they tend t o be i s o l a t e d , or both f a c t o r s may c o n t r i b u t e t o low h e t e r o z y g o s i t y . R e l a t i o n s h i p s Between Benthic And Limnetic S t i c k l e b a c k s The m o r p h o l o g i c a l l y and e c o l o g i c a l l y d i s t i n c t b e n t h i c and l i m n e t i c s t i c k l e b a c k s o f Paxton and Enos l a k e s d i s p l a y e d s t r i k i n g l y d i v e r g e n t enzyme patterns..,For both l a k e s , G t e s t s i n d i c a t e d t h a t a l l e l e f r e q u e n c i e s at the Ck, Pgm and Mdh-3 l o c i d i f f e r e d s i g n i f i c a n t l y between b e n t h i c and l i m n e t i c p o p u l a t i o n s , and i n Paxton Lake s i g n i f i c a n t d i f f e r e n c e s i n a l l e l e f r e q u e n c i e s a l s o e x i s t e d a t the Pgi-2 lo c u s (Table 12) . This i n d i c a t e s t h a t i n each l a k e the be n t h i c and l i m n e t i c s t i c k l e b a c k s comprise g e n e t i c a l l y d i s t i n c t p o p u l a t i o n s . The g e n e t i c d i s t a n c e between b e n t h i c and l i m n e t i c s t i c k l e b a c k s i n Paxton Lake was 0.1136, and i n Enos Lake was 0,1263. These d i s t a n c e s a re twice as l a r g e as 0.0632, the average g e n e t i c d i s t a n c e between freshwater p o p u l a t i o n s . S t i c k l e b a c k s i n Cranby, Hiechhold and Goose la k e s were a l s o polymorphic at t h e Ck and Pgm l o c i , and i n Hiechhold 77 Table 12. , A l l e l e frequency v a r i a b i l i t y between b e n t h i c and l i m n e t i c s t i c k l e b a c k s . G values were c a l c u l a t e d f o r a l l e l e d i s t r i b u t i o n s at polymorphic l o c i . . The L i t t l e Campbell comparison i s betwwen the l e i u r u s and trachurus p o p u l a t i o n s . C r i t i c a l c h i - s q u a r e values at .05 l e v e l are i n b r a c k e t s . , Locus Paxton Enos L. ..Campbell Lake* L a k e 2 fiiver3 Obs.,G Value: 0.35 0.27 Mdh-1 C r i t . Chi-square: (3.84) (3.84) Obs. G V a l u e : 51.66*** 69.18*** 42.97*** Mdh-3 C r i t . Chi-square: (3.84) (3.84) (3.84) Obs. G Value: 50.27*** 1.32 3.70 Pgi-2 C r i t . Chi-square: (3.84) (3.84) (3.84) Obs.,G Value: 144.81*** 172.48*** 146.00*** Pgm C r i t . C h i -square: (5.99) (5-99) (5.99) Obs. „G Value: 80.02*** 5 5 4 . 3 0 * * * 164.76*** Ck C r i t. Chi -squ ar e: (3.84) (3.84) (3.84) Obs. G Value: 22. 67*** 21.14*** 11.13** Idh C r i t . C h i -square: (3.84) (3.84) (3.84) ** p < 0.001 #** p < 0.0001 » 1977 samples « combined 1977 and 1978 samples 3 1978 samples 78 and Goose l a k e s a t the Mdh-3 and Pgi-2 l o c i as v e i l . A l l e l e frequency d i f f e r e n c e s between phenotypes were not examined i n these l a k e s . M o r p h o l o g i c a l l y d i s t i n c t p o p u l a t i o n s of s t i c k l e b a c k s a l s o come i n contact i n many streams i n which both t r a c h u r u s and l e i u r u s p o p u l a t i o n s breed i n the s p r i n g and e a r l y summer. In the L i t t l e Campbell B i v e r , the breeding r e g i o n s o v e r l a p and some i n t e r b r e e d i n g a p p a r e n t l y occurs (Hagen 1967)., M o r p h o l o g i c a l l y , the t r a c h u r u s bear a c l o s e r resemblance t o the l i m n e t i c than to th e b e n t h i c s t i c k l e b a c k s of Paxton and Enos l a k e s . They a r e t e r e t e and h e a v i l y armoured, and possess the long and numerous g i l l r a k e r s and s i l v e r y c o u n t e r - c o l o u r a t i o n c h a r a c t e r i s t i c of f i s h l e a d i n g a p e l a g i c e x i s t e n c e . The L i t t l e Campbell l e i u r u s , on the other hand, resemble the l a c u s t r i n e b e n t h i c s t i c k l e b a c k s , both i n t h e i r deep body shape and reduced armour as w e l l as i n dark c o l o u r a t i o n . The amount and nature of the g e n e t i c d i f f e r e n t i a t i o n between the l e i u r u s and t r a c h u r u s s t i c k l e b a c k s p o p u l a t i o n s was very s i m i l a r to that between the l a c u s t r i n e b e n t h i c s and l i m n e t i c s . A l l e l e f r e q u e n c i e s at the Ck, Pgm and Mdh-3 l o c i d i f f e r e d s i g n i f i c a n t l y between the two types (Table 12) , and Pgi-2 was s l i g h t l y polymorphic i n the marine but not the f r e s h w a t e r p o p u l a t i o n . The g e n e t i c d i s t a n c e between the l e i u r u s and t r a c h u r u s p o p u l a t i o n s was 0.1006, g r e a t e r than the average d i s t a n c e of 0.0608 between marine and freshwater p o p u l a t i o n s . F i g u r e 6 i l l u s t r a t e s t h a t i n each of the p a i r s of p o p u l a t i o n s , not only were the same l o c i polymorphic and d i f f e r e n t between the two morphs, but the d i f f e r e n c e s i n a l l e l e 79 F i g u r e 6. A l l e l e frequency d i f f e r e n t i a t i o n between b e n t h i c and l i m n e t i c s t i c k l e b a c k s . The L i t t l e Campbell comparison i n v o l v e s the t r a c h u r u s ( l i m n e t i c ) and l e i u r u s (benthic) p o p u l a t i o n s . 7?a PAXTON ENOS L. CAMPBELL LAKE LAKE RIVER 1.0 CK 1 0 0 FREQUENCY 0 5 o.o 1.0 i M d h - 3 5 5 FREQUENCY o.s-I 0.0 1.0 - i P g m 1 0 3 FREQUENCY o.s 1.0 - i P g m 9 0 F R E Q U E N C Y * M o.o i — i BENTHIC L IMNETIC • 80 f r e q u e n c i e s were i n the same d i r e c t i o n , Thus, the frequency of the C k 1 0 0 a l l e l e was higher i n a l l t h r e e benthic p o p u l a t i o n s , t h e freguency of Mdh-3 S S was higher i n a l l t h r e e l i m n e t i c p o p u l a t i o n s , the Pgm 1 0 3 a l l e l e was more common i n b e n t h i c and Pgm»o i n l i m n e t i c p o p u l a t i o n s . H h i l e the average genetic d i s t a n c e between each of the b e n t h i c - l i m n e t i c p a i r s was 0.1135, the average d i s t a n c e between the t h r e e benthic p o p u l a t i o n s was 0.0275 and between the three l i m n e t i c p o p u l a t i o n s was 0.0263. 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 survey of G. a c u l e a t u s p o p u l a t i o n s i n d i c a t e s the morphological v a r i a b i l i t y c h a r a c t e r i s t i c of t h i s s p e c i e s i s accompanied by c o n s i d e r a b l e h e t e r o g e n e i t y at the molecular l e v e l . S i x of e i g h t enzyme l o c i examined e x h i b i t e d g e n e t i c a l l y c o n t r o l l e d v a r i a b i l i t y i n isozyme banding p a t t e r n s . Although e i g h t i s a s m a l l number of l o c i on which t o base est i m a t e s of polymorphism and h e t e r o z y g o s i t y f o r use i n i n t e r s p e c i f i c comparisons, the primary purpose of t h i s study was to examine i n t r a s p e c i f i c r e l a t i o n s h i p s . With the e x c e p t i o n of c r e a t i n e k i n a s e , which had been s t u d i e d p r e v i o u s l y i n Gasterosteus JHagen 1967, B i c h i e l 1977), none of the l o c i examined were known t o be polymorphic when the study began. The remaining seven enzymes were chosen by v i r t u e of c l e a r r e s o l u t i o n of the isozyme bands, and the subsequent c o n f i r m a t i o n of t h e i r g e n e t i c c o n t r o l . Thus, the e s t i m a t e s of g e n e t i c v a r i a b i l i t y d e r i v e d from gene f r e q u e n c i e s at these l o c i a r e unbiased by conscious s e l e c t i o n f o r h e t e r o g e n e i t y . N e v e r t h e l e s s , a t l e a s t three of the enzymes used i n t h e present 81 study are n o t a b l y polymorphic i n f i s h s p e c i e s : CK ( F e r r i s and Whitt 1978), PGI (Avise and K i t t o 1973) and PGM. T h e r e f o r e , a d i s p r o p o r t i o n a t e number of polymorphic l o c i may be i n c l u d e d . , In a r e c e n t study, Avise (1976) examined v a r i a b i l i t y a t as many a s 15 l o c i i n three C a l i f o r n i a G a sterosteus p o p u l a t i o n s . In a d d i t i o n t o polymorphism a t l o c i examined i n t h i s study (Mdh-1, Hdh-3 (his Hdh-2), Pgi-2, Pgm and Ck (his Pt-3?)) , he r e p o r t e d v a r i a b i l i t y at an e s t e r a s e l o c u s . E s t - 1 , a t r i o s e p h o s p h a t e isomerase l o c u s , T p i - 1 , and a g e n e r a l p r o t e i n l o c u s , P t - 1 . The i n c l u s i o n of a l a r g e p r o p o r t i o n of a t y p i c a l l y v a r i a b l e l o c i accounts, a t l e a s t i n p a r t , f o r the high average values of h e t e r o z y g o s i t y and polymorphism i n d i c a t e d by the data. The values of 0.124 f o r h e t e r o z y g o s i t y and 0.466 f o r polymorphism are c o n s i d e r a b l y g r e a t e r than the mean values of 0.051 and 0.152 r e s p e c t i v e l y , c a l c u l a t e d f o r 51 s p e c i e s of O s t e i c h t h y e s (Nevo 1978). In t h a t review, t h e only s p e c i e s with v a l u e s exceeding those of the p r e s e n t study was the k i l l i f i s h , Fundulus h e t e r o c l i t u s ( H i t t o n and Koehn 1975). C o n s i d e r a b l y lower e s t i m a t e s of both h e t e r o z y g o s i t y (0.09) and polymorphism (0.27) f o r Gasterosteus were o b t a i n e d by Avise (1976), and although based on samples from a s i n g l e p o p u l a t i o n * were d e r i v e d from i n f o r m a t i o n a t 15 g e n e t i c l o c i . , No d e v i a t i o n s from Hardy-Weinberg c o n d i t i o n s were found i n t h e genotypic d i s t r i b u t i o n s a t any polymorphic l o c u s beyond those a t t r i b u t a b l e t o sampling e r r o r . C l o s e agreement between observed and expected genotypic p r o p o r t i o n s i s the r u l e r a t h e r than the e x c e p t i o n i n analyses o f e l e c t r o p h o r e t i c gene f r e q u e n c i e s , and more l i k e l y r e f l e c t s the weakness of the t e s t 82 (Workman 1969, Hard and S i n g 1970) r a t h e r than p r e c l u d e s the p o s s i b i l i t y of s e l e c t i o n or non-random breeding w i t h i n the examined p o p u l a t i o n s . In a d e t a i l e d study of an e e l p o u t (Zoarces v i v i p a r u s l p o p u l a t i o n , C h r i s t i a n s e n e t a l . (1973) found that p o s t - z y g o t i c s e l e c t i o n favoured E s t - I I I homozygotes a t the expense of i n d i v i d u a l s heterozygous a t t h a t l o c u s . Although no s i g n i f i c a n t d e v i a t i o n from Hardy-Weinberg c o n d i t i o n s was apparent, there was a s i g n i f i c a n t d e f i c i e n c y of E s t - I I I h e t e r o z y g o t e s r e v e a l e d among the a d u l t e e l p o u t p o p u l a t i o n by more powerful methods of m o t h e r - o f f s p r i n g a n a l y s i s , and l a t e r by age group a n a l y s i s ( C h r i s t i a n s e n et a l . 1974).. The observed s t a b i l i t y o f gene f r e g u e n c i e s over s h o r t time p e r i o d s i n t h i s study i s a l s o c h a r a c t e r i s t i c of other s t u d i e s on f i s h p o p u l a t i o n s ( A l l e n d o r f and Otter 1979, Avise and F e l l e y 1979) to the l i m i t e d extent t h a t temporal v a r i a b i l i t y has been examined. However, s t u d i e s with other organisms (Gaines et a l . 1978, Berger 1971, Dobzhansky and Ayala 1973) have i n d i c a t e d t h a t temporal g e n e t i c v a r i a b i l i t y , mediated by s e l e c t i o n , does occur w i t h i n p o p u l a t i o n s . , The change i n the frequency of the Mdh-3»°° a l l e l e i n the Serpentine River (TYHEP) from 0.26 i n March 1978 t o 0.06 i n June 1979 may r e f l e c t such a process, but c o n f i r m a t i o n would r e q u i r e i n v e s t i g a t i o n of the p o p u l a t i o n s t r u c t u r e and the i d e n t i f i c a t i o n of the s e l e c t i v e agent (s). Small d i f f e r e n c e s i n a l l e l e f r e q u e n c i e s at the Mdh-3 and Ck l o c i between 1977 and 1978 samples of Enos Lake l i m n e t i c s may r e f l e c t r e a l changes i n the g e n e t i c composition of the p o p u l a t i o n , but more l i k e l y r e s u l t e d from the i n c l u s i o n of b e n t h i c or h y b r i d f i s h i n the 1977 sample. In that year, f i s h 83 were not sep a r a t e d on a mor p h o l o g i c a l b a s i s before e l e c t r o p h o r e s i s , but merely by method of capture (minnow t r a p or ni g h t - t i m e seine haul) and some m i s c l a s s i f i c a t i o n l i k e l y r e s u l t e d . F a i l u r e to r e c o g n i z e gene frequency changes i n other p o p u l a t i o n s may r e f l e c t the s h o r t time i n t e r v a l s between sample c o l l e c t i o n s , or sample s i z e s too s m a l l t o r e v e a l the s i g n i f i c a n c e of s l i g h t , but r e a l , changes. Lack o f s t a n d a r d i z a t i o n of the age and sex composition of repeated samples may a l s o obscure r e a l s h i f t s i n gene f r e q u e n c i e s that occur over time. While the d i s p r o p o r t i o n a t e number of polymorphic l o c i i n c l u d e d i n my study hinders comparisons of g e n e t i c v a r i a b i l i t y with other s p e c i e s , i t f a c i l i t a t e s examination of r e l a t i o n s h i p s among c o n s p e c i f i c p o p u l a t i o n s * Both monomorphic l o c i , Ldh and Idh, are f i x e d f o r the same a l l e l e i n a l l p o p u l a t i o n s , marine and freshwater, and provide no i n f o r m a t i o n on, the g e n e t i c r e l a t i o n s h i p of any p o p u l a t i o n t o another. The degree of d i f f e r e n t i a t i o n a t polymorphic l o c i , on the other hand, can be used as a measure o f ge n e t i c d i s t a n c e between p o p u l a t i o n s , and from such data e v o l u t i o n a r y i n f e r e n c e s are of t e n drawn^ Nei's g e n e t i c d i s t a n c e , D, c o n s t i t u t e s a measure of the accumulated number o f gene s u b s t i t u t i o n s per l o c u s between two p o p u l a t i o n s , which, i f the r a t e of gene s u b s t i t u t i o n per year i s c o n s t a n t ( i . e . ..time-dependent), i s l i n e a r l y r e l a t e d to e v o l u t i o n a r y time (Nei 1972, 1975) . I n c r e a s i n g evidence f o r the s e l e c t i v e value of some 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 demonstrates c o n v i n c i n g l y t h a t h e t e r o g e n e i t y at t h e enzyme l e v e l i s not a l l 84 s e l e c t i v e l y n e u t r a l , nor c o n t r o l l e d e n t i r e l y by p o p u l a t i o n parameters and s t o c h a s t i c events over time. S i g n i f i c a n t c o r r e l a t i o n s between enzyme genotypes and environmental v a r i a b l e s (such as temperature) are known f o r a number o f f i s h s p e c i e s (Sick 1965, Koehn and Easmussen 1967, Johnson 1971, Frydenberg et a l . , 1973, Nyman 1975, a i t t o n and Koehn 1975) as w e l l as f o r many other organisms r a n g i n g from s n a i l s (Johnson 1976) to ants (Tomaszewski e t a l . 1973) t o b a r l e y (Hamrick and A l l a r d 1975) . In some cases, f u n c t i o n a l c h a r a c t e r i s t i c s of the d i f f e r e n t a l l e l e s at a l o c u s have been examined, and the optimal performance c o n d i t i o n s of each a l l e l i c product found to correspond t o the n a t u r a l environment i n which the a l l e l e commonly occurs ( H e r r i t t 1972, Koehn 1969)..To the extent that 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 s u b j e c t t o n a t u r a l s e l e c t i o n , measures of g e n e t i c d i s t a n c e p r o v i d e good e s t i m a t e s of the g e n e t i c d i f f e r e n t i a t i o n among p o p u l a t i o n s , but not o f t h e i r e v o l u t i o n a r y r e l a t i o n s h i p s . . In the present study, r e l a t i o n s h i p s among and between marine and freshwater s t i c k l e b a c k s are examined on the b a s i s of g e n e t i c d i s t a n c e . B e l l (1976) p o s t u l a t e d t h a t the freshwater p o p u l a t i o n s i n r e g i o n s i n c l u d i n g the present study area are the r e s u l t of p o s t g l a c i a l p o l y p h y l e t i c e v o l u t i o n from the marine t r a c h u r u s form.. He thus suggested t h a t e l e c t r o p h o r e t i c genetic h e t e r o g e n e i t y should be g r e a t e r w i t h i n trachurus than l e i u r u s p o p u l a t i o n s , owing to t h e i r g r e a t e r age and l e s s e r v u l n e r a b i l i t y t o the e r o s i o n of g e n e t i c v a r i a b i l i t y through founder e f f e c t s and g e n e t i c d r i f t . On the other hand, g e n e t i c h e t e r o g e n e i t y between the i n t e r c o n n e c t e d marine p o p u l a t i o n s should be l e s s 85 than between those occupying d i s j u n c t freshwater l o c a l i t i e s . Thus, while marine p o p u l a t i o n s should be g e n e t i c a l l y s i m i l a r by v i r t u e of gene flow, the independently e s t a b l i s h e d freshwater p o p u l a t i o n s s h o u l d bear no g r e a t e r resemblance t o each other than t o the marine p o p u l a t i o n s from which they descended.. Although there i s no way o f e s t a b l i s h i n g the degree of s i m i l a r i t y between gene f r e g u e n c i e s i n extant marine p o p u l a t i o n s and those c h a r a c t e r i z i n g p o s t g l a c i a l marine p o p u l a t i o n s , the present nature and degree 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 i n marine s t i c k l e b a c k s i s compatible with the i d e a that a l l freshwater p o p u l a t i o n s have a marine o r i g i n . / The marine p o p u l a t i o n s possess ( u s u a l l y a t low freguencies) a l l the e l e c t r o p h o r e t i c v a r i a n t s d e t e c t e d i n freshwater p o p u l a t i o n s . Even the band d e f i n e d by Hagen (1967) as " d i a g n o s t i c " f o r the marine form i n the L i t t l e Campbell B i v e r has been found i n l a k e p o p u l a t i o n s (unpub. data) ; however, i t does seem t o be absent from r e s i d e n t stream-dwelling p o p u l a t i o n s such as the one Hagen examined. In a d d i t i o n , the r e s u l t s of the present study confirm a number of B e l l ' s (1976) p r e d i c t i o n s . The average g e n e t i c d i s t a n c e between marine p o p u l a t i o n s i s 0.0055 while between freshwater p o p u l a t i o n s i t i s 0.0632. Thus, there i s over t e n times more d i s t a n c e between freshwater than between marine p o p u l a t i o n s . , As p r e d i c t e d , the s m a l l e r d i s t a n c e s between marine p o p u l a t i o n s i n d i c a t e t h a t they are g e n e t i c a l l y more homogeneous than freshwater p o p u l a t i o n s . Moreover, the average d i s t a n c e between marine and freshwater p o p u l a t i o n s i s s l i g h t l y l e s s than the average between p a i r s of freshwater p o p u l a t i o n s (0.0608). 86 Again, as p r e d i c t e d , freshwater p o p u l a t i o n s are no more s i m i l a r t o one another than they are t o marine p o p u l a t i o n s . The average number of polymorphic l o c i i n marine p o p u l a t i o n s i s 4.8 (60%), and i n freshwater p o p u l a t i o n s i t i s 3.4 (13%). T h i s supports the c o n t e n t i o n that i n t r a p o p u l a t i o n h e t e r o g e n e i t y s h o u l d be grea t e r w i t h i n marine than w i t h i n freshwater p o p u l a t i o n s . In c o n t r a s t , however, average h e t e r o z y g o s i t y i s s l i g h t l y higher i n freshwater (0.126) than i n marine (0.118) p o p u l a t i o n s . Thus, while the marine environment possesses the e n t i r e range o f e l e c t r o p h o r e t i c v a r i a n t s , these v a r i a n t s occur a t uniformly low , f r e g u e n c i e s i n marine p o p u l a t i o n s . These r e s u l t s concerning the i n t r a - and i n t e r p o p u l a t i o n v a r i a b i l i t y i n marine and freshwater p o p u l a t i o n s can a l s o be e x p l a i n e d i n another way. I f the tenuous assumption of s e l e c t i v e n e u t r a l i t y made by B e l l (1976) i s d i s c a r d e d , measures of genetic d i s t a n c e are no longer n e c e s s a r i l y r e l a t e d t o e v o l u t i o n a r y time i n a l i n e a r f a s h i o n . In Ga s t e r o s t e u s , the gr e a t e r 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 among freshwater p o p u l a t i o n s may r e s u l t from the l a r g e v a r i e t y o f s e l e c t i v e regimes imposed by d i v e r s e freshwater environments and the absence of gene flow among these d i s j u n c t p o p u l a t i o n s . The e l e c t r o p h o r e t i c homogeneity among marine s t i c k l e b a c k p o p u l a t i o n s may r e s u l t from the r e l a t i v e u n i f o r m i t y o f marine environments combined with the p o s s i b i l i t y of gene flow among them. Thus, the l a r g e g e n e t i c d i s t a n c e s among freshwater p o p u l a t i o n s , and between freshwater and marine p o p u l a t i o n s , may r e f l e c t the l a r g e d i f f e r e n c e s i n s e l e c t i v e f o r c e s among these environments r a t h e r than the long 87 e v o l u t i o n a r y i s o l a t i o n of p o p u l a t i o n s occupying them. A comparison of a l l e l i c d i f f e r e n t i a t i o n between the Somass (west coast of Vancouver Island) and Bear (east coast) r i v e r systems r e v e a l s t h a t n e i t h e r drainage system nor h a b i t a t type accounts f o r a l a r g e amount of the great among po p u l a t i o n v a r i a b i l i t y i n a l l e l e f r e q u e n c i e s found i n the 19 l o c a t i o n s sampled.. Only the frequency of Mdh-1 l°o i s s i g n i f i c a n t l y heterogeneous among h a b i t a t s . T h i s i s due to r e l a t i v e l y high f r e q u e n c i e s of the v a r i a n t a l l e l e Mdh-1 8 2 i n l a r g e l a k e s as compared t o smal l l a k e s . , N e v e r t h e l e s s , g e n e t i c d i s t a n c e s are s l i g h t l y l e s s between p o p u l a t i o n s from the same drainage (Bear or Somass) (0.038), than between p o p u l a t i o n s from d i f f e r e n t d rainages (0.047). This p r o v i d e s some evidence f o r i n t r a d r a i n a g e homogeneity; however, the degree of g e n e t i c d i s t i n c t n e s s of Ga s t e r o s t e u s w i t h i n the two watersheds was much l e s s than found by Hedgecock (1978) i n a s i m i l a r comparison between watersheds of salamander ( T a r i c h a r i v n l a r i s) p o p u l a t i o n s . , For Tar i c h a , the average between drainage g e n e t i c d i s t a n c e , a l t h o u g h l e s s than both t h e between and w i t h i n drainage d i s t a n c e s f o r s t i c k l e b a c k s , was almost f o u r times as gre a t as the average w i t h i n drainage d i s t a n c e . In c o n t r a s t , r e s u l t s s i m i l a r t o mine were r e p o r t e d by Avise and Smith (1974) and Avise and F e l l e y (1979) f o r sout h e a s t e r n U. S. r e s e r v o i r p o p u l a t i o n s of b l u e g i l l (lepomis macrochirus). W i t h i n macrogeographic a r e a s , these i n v e s t i g a t o r s found r e l a t i v e homogeneity i n a l l e l e f r e q u e n c i e s at polymorphic l o c i within p o p u l a t i o n s ( r e s e r v o i r s ) , and g r e a t g e n e t i c h e t e r o g e n e i t y among r e s e r v o i r s w i t h i n a drainage system. There was no s i g n i f i c a n t 88 i n c r e a s e i n a l l e l e frequency variance i n comparisons between drainage systems (Avise and Smith 1974). There was, however, g e n e t i c d i f f e r e n t i a t i o n between drainages i n t h r e e d i f f e r e n t macrogeographic r e g i o n s i n h a b i t e d by two separate subspecies of L. macrochirus and t h e i r h y b r i d s . While the l a c k of g e n e t i c d i s t i n c t i o n between the Somass and Bear r i v e r systems f a i l s t o provide c o n v i n c i n g support f o r the s u g g e s t i o n of independent e v o l u t i o n w i t h i n d r a i n a g e s , data from a l a r g e r number of more g e o g r a p h i c a l l y widespread r i v e r systems are r e g u i r e d to r i g o r o u s l y t e s t the hypothesis. , l t might be argued t h a t the c l o s e p r o x i m i t y of the two watersheds i n t h i s study i n d i c a t e s a common p o s t g l a c i a l h i s t o r y , with a high p r o b a b i l i t y o f simultaneous c o l o n i z a t i o n by c l o s e l y r e l a t e d marine p o p u l a t i o n s . The f a c t t h a t even the between drainage g e n e t i c d i s t a n c e of 0.047 i s l e s s than the average d i s t a n c e of 0.063 between a l l freshwater p o p u l a t i o n s supports t h i s s uggestion. However, Mathews et a l . (1970) suggest t h a t marine i n v a s i o n i n the A l b e r n i i n l e t on the west coa s t ( i n t o which the Somass R i v e r flows) may have occurred s i g n i f i c a n t l y before i n v a s i o n of the e a s t e r n s i d e of Vancouver I s l a n d (on which t h e Bear system l i e s ) . The r e t r e a t of g l a c i a l i c e i n an e a s t e r l y d i r e c t i o n i s r e s p o n s i b l e f o r the d i f f e r e n c e i n t i m i n g . , Even i f the amount of g e n e t i c v a r i a t i o n between watersheds i s g e n e r a l l y g r e a t e r than t h a t d i s t i n g u i s h i n g the Bear and Somass systems, t h e r e o b v i o u s l y e x i s t s another s o u r c e of v a r i a b i l i t y u n d e r l y i n g t h e l a r g e amounts of genie h e t e r o g e n e i t y observed between l o c a l i t i e s w i t h i n a s i n g l e d r a i n a g e . „. I t i s p o s s i b l e that e l e c t r o p h o r e t i c d i f f e r e n t i a t i o n w i t h i n , as well as 89 between, watersheds i s a r e f l e c t i o n of founder e f f e c t s and g e n e t i c d r i f t . G e o l o g i c a l evidence i n d i c a t e s t h a t with the r e t r e a t of the l a s t major g l a c i a t i o n from Vancouver I s l a n d and the lower Fraser V a l l e y {approximately 13,000 years ago) land l e v e l s rose r a p i d l y . .Mathews e t a l . (1970) s t a t e : "... the f i r s t 300 f t of emergence (out of an ultimate u p l i f t of perhaps 700 f t ) occurred w i t h i n a few hundred years, and the f i r s t 500 f t i n not more than 1,000 years"., At present, n a t u r a l p o p u l a t i o n s of s t i c k l e b a c k s on Vancouver I s l a n d and i n the F r a s e r V a l l e y occur on l y a t e l e v a t i o n s up t o 700 f t ( J . D. McPhail, p e r s . comm.), and maximum e l e v a t i o n s decrease on s o u t h e r n Vancouver I s l a n d where Mathews et a l . (1970) i n d i c a t e p o s t g l a c i a l l a n d emergence was l e a s t . A subsequent, l e s s severe submergence of l a n d , and accompanying marine t r a n s g r e s s i o n of t e r r e s t r i a l h a b i t a t s , o c c u r r e d d u r i n g a minor g l a c i a l advance approximately 11,000 years ago. T h i s p r o v i d e d an o p p o r t u n i t y f o r secondary marine i n v a s i o n a t lower e l e v a t i o n s . The f o l l o w i n g re-emergence of land t o l e v e l s not g r e a t l y d i f f e r e n t from those of today was complete about 9,000 years ago. L e v e l s have v a r i e d l i t t l e (about 35 f t ) s i n c e t h a t time (Mathews e t a l . 1970) . Small numbers of the founding marine p o p u l a t i o n s , or t h e i r e a r l y descendants, were l i k e l y i s o l a t e d i n s u i t a b l e h a b i t a t s w i t h i n drainages as p o s t g l a c i a l l a n d l e v e l s rose and sea l e v e l s dropped. The s i g n i f i c a n t c o r r e l a t i o n between geographic and g e n e t i c d i s t a n c e w i t h i n the Bear system may be the r e s u l t of r e s t r i c t e d gene flow between adj a c e n t l o c a l i t i e s as water l e v e l s subsided. . C e r t a i n l y the s m a l l l a k e s at higher e l e v a t i o n s were 90 the f i r s t t o be i s o l a t e d , and the low l e v e l s of h e t e r o z y g o s i t y i n the headwater l a k e s o f the Bear drainage a r e c o n s i s t e n t with the concept t h a t gene flow a t that time a f f e c t e d t h e present g e n e t i c s t r u c t u r e of p o p u l a t i o n s . L a r g e r founding p o p u l a t i o n s , g r e a t e r gene flow among them and, p o s s i b l y , secondary i n v a s i o n by marine p o p u l a t i o n s , may a l l have c o n t r i b u t e d t o the present high l e v e l s of h e t e r o z y g o s i t y i n the l a r g e l a k e s and l o w - l y i n g streams occupying the lower reaches of watersheds. The present s t r i k i n g d i f f e r e n t i a t i o n among p o p u l a t i o n s w i t h i n drainages may be the r e s u l t of genetic d r i f t over the past 9,000 years (while sea l e v e l s have been st a b l e ) compounding the o r i g i n a l founder e f f e c t s among l o c a l i t i e s . C e r t a i n l y , extreme morphological d i f f e r e n t i a t i o n over s h o r t d i s t a n c e s a t t e s t s s t r o n g l y to the e f f e c t i v e n e s s o f r e g i o n s of poor h a b i t a t as b a r r i e r s t o present day gene flow.. Thus the observed p a t t e r n s i n h e t e r o z y g o s i t y l e v e l s can be a t t r i b u t e d t o h i s t o r i c a l events a f f e c t i n g gene flow w i t h i n and between p o p u l a t i o n s . M e r r i t t e t a l . (1978) a t t r i b u t e d a s i m i l a r c l i n e i n l e v e l of h e t e r o z y g o s i t y i n longnose dace ( S h i a j c h t h y s c a t a r a c t a e ) p o p u l a t i o n s of the South Connecticut B i v e r t o t h e s t o c h a s t i c processes of founder e f f e c t and d r i f t . A v i s e and F e l l e y (1979) a l s o emphasized the importance of breeding s t r u c t u r e and gene flow i n t h e i r study of i n t r a d r a i n a g e 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 i n the b l u e g i l l . However, they noted: "... i t may not be unreasonable t o propose t h a t s e l e c t i o n d i f f e r e n t i a l s between r e s e r v o i r s are f a r grea t e r than those w i t h i n , p a r t i c u l a r l y s i n c e roughly p a r a l l e l c l i n e s of a l l e l e f r e g u e n c i e s occur across t h e s e two p r e s e n t l y i s o l a t e d 91 d r a i n a g e s " . Convincing arguments can a l s o be made f o r the r o l e of s e l e c t i o n i n b r i n g i n g about the p a t t e r n s of g e n e t i c v a r i a b i l i t y d i s p l a y e d by Gasterosteus p o p u l a t i o n s . Low l e v e l s of h e t e r o z y g o s i t y are found t o c h a r a c t e r i z e not only the s m a l l lake p o p u l a t i o n s of the Bear and Somass systems, but the p o p u l a t i o n s of small l a k e s and i s o l a t e d streams i n g e n e r a l . The two most l i k e l y e x p l a n a t i o n s f o r t h i s l a c k o f genetic v a r i a b i l i t y are (1) founder e f f e c t s , and g e n e t i c d r i f t i n s m a l l post-founding p o p u l a t i o n s or during p o p u l a t i o n b o t t l e n e c k s , as d i s c u s s e d above, or (2) d i r e c t i o n a l s e l e c t i o n imposed by homogeneous environmental c o n d i t i o n s ( i . e . , l a c k of niche v a r i a b l i t y ) . Founder e f f e c t s and g e n e t i c d r i f t have l i k e l y a f f e c t e d the t i n y p o p u l a t i o n s of i s o l a t e d streams and swamps. These occupy h a b i t a t s l e s s than 0.01 km2 i n a r e a , and are c o n t i n u a l l y s u b j e c t e d to environmental s t r e s s (such as winter f r e e z i n g and summer drought). P o p u l a t i o n l e v e l s l i k e l y range between 10 2 and 5 x 10 3 during s p r i n g and summer breeding, but overwinter s u r v i v a l i s minimal. Small l a k e s , on the other hand, are g e n e r a l l y much l a r g e r (up to 1 km 2 i n t h i s study) and p r o v i d e c o n s i d e r a b l e b u f f e r i n g from c l i m a t i c extremes. P o p u l a t i o n l e v e l s reach 10* t o 10 s or more durin g summer months, and, although winter m o r t a l i t y may be h i g h , e s p e c i a l l y among a d u l t s , p o p u l a t i o n l e v e l s probably do not drop t o those at which g e n e t i c d r i f t becomes important, Mark and rec a p t u r e e s t i m a t e s of p o p u l a t i o n s i z e of the i n t r o d u c e d Marion Lake s t i c k l e b a c k s i n d i c a t e d that the 4,000 s t i c k l e b a c k s planted 92 i n the summer of 1974 had i n c r e a s e d to 60,000 i n the summer of 1975, to over 100,000 i n the summer of 1976 and have averaged around 60,000 i n subsequent years. I t appears u n l i k e l y , t h e n , t h a t q e n e t i c d r i f t c o n s t i t u t e s the mechanism r e s p o n s i b l e f o r reduced 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 i n s m a l l - l a k e G a s t e r o s t e u s p o p u l a t i o n s . although founder e f f e c t s cannot be d i s m i s s e d as a p o s s i b l e f a c t o r , the l a r g e amounts of m o r p h o l o g i c a l d i f f e r e n t i a t i o n t h a t o c c u r r e d i n the process of d e r i v i n g present day freshwater p o p u l a t i o n s from the o r i g i n a l marine form i n d i c a t e t h a t l e v e l s o f g e n e t i c v a r i a b i l i t y i n founding p o p u l a t i o n s were not extremely low. . Gorman et a l . (1975) working on i s l a n d p o p u l a t i o n s of a d r i a t i c l i z a r d s c a l c u l a t e d t h a t a s i n g l e pregnant female would i n t r o d u c e 34% of the s p e c i e s v a r i a b i l i t y i n t o a new p o p u l a t i o n . T h i s i l l u s t r a t e s the very s m a l l s i z e s of founding p o p u l a t i o n s r e q u i r e d f o r founder e f f e c t s to be pronounced. The s t r u c t u r a l s i m i l a r i t y i n h a b i t a t s occupied by i s l a n d p o p u l a t i o n s of t e r r e s t r i a l organisms and lake p o p u l a t i o n s of freshwater organisms was noted by Avise and Smith (1974). These authors suggested t h a t the g e n e t i c d i s t i n c t i o n of lake p o p u l a t i o n s , l i k e t h a t of i s l a n d p o p u l a t i o n s , i s the r e s u l t of i n c r e a s e d g e n e t i c d r i f t under c o n d i t i o n s of geographic i s o l a t i o n imposed by the p h y s i c a l b a r r i e r s of l a n d , i n the case of l a k e s , and water, i n the case of i s l a n d s . S t i c k l e b a c k s , occupying both t h e ocean and i s o l a t e d freshwater h a b i t a t s , are d i s t r i b u t e d remarkably l i k e those t e r r e s t r i a l organisms that populate both mainland and i s l a n d s i t e s . 93 Gorman et a l . (1975) found t h a t p a t t e r n s i n h e t e r o z y g o s i t y l e v e l s s i m i l a r t o those of the presen t study c h a r a c t e r i z e d mainland and i s l a n d p o p u l a t i o n s of the l i z a r d L a c e r t a s i c u l a . These l i z a r d s possess a number of f e a t u r e s i n common with G a s t e r o s t e u s . They e x h i b i t broad e c o l o g i c a l t o l e r a n c e , and are both p o l y t y p i c ( d i s p l a y i n g s t r i k i n g v a r i a b i l i t y i n c o l o u r and morphology between populations) and polymorphic (possessing w i t h i n p o p u l a t i o n v a r i a b i l i t y f o r a number of morphological t r a i t s ) I s l a n d (the t e r r e s t r i a l e q u i v a l e n t of s m a l l lake) p o p u l a t i o n s possessed lower l e v e l s of h e t e r o z y g o s i t y than d i d mainland ( t e r r e s t r i a l l y e q u i v a l e n t t o marine) p o p u l a t i o n s . The l i z a r d s on very small i s l a n d s {< 0.01 km 2), l i k e t he s t i c k l e b a c k s of i s o l a t e d streams and swamps, were even l e s s heterozygous. As i n the present study, t h i s " s m a l l i s l a n d e f f e c t " i n the s m a l l e s t of p o p u l a t i o n s was a t t r i b u t e d , at l e a s t i n p a r t , t o g e n e t i c d r i f t and founder e f f e c t s . Gorman e t a l . (1975) noted, however, that "although g e n e t i c d r i f t might account f o r the p o s s i b l e l o s s of a l l e l e s i n the s m a l l e s t f r i n g i n g p o p u l a t i o n s , t h i s i n no way i m p l i e s that t h e a l l e l e s are behaving n e u t r a l l y " . On other i s l a n d s (> 0.05 km2) Gorman e t a l . , a t t r i b u t e d the hi g h e r , but s t i l l low l e v e l s of h e t e r o z y g o s i t y t o s e l e c t i o n . They proposed the "time-divergence" hypothesis t o e x p l a i n t h e i r r e s u l t s (Soule and Yang 1973, Gorman et a l . 1975). S p e c i f i c a l l y , they f e l t t h a t "(1) ge n e t i c v a r i a b i l i t y i s l o s t as a conseguence o f d i r e c t i o n a l s e l e c t i o n at r a t e s p r o p o r t i o n a l to average e v o l u t i o n a r y r a t e s and (2) e v o l u t i o n a r y r a t e s of i s l a n d 94 r e p t i l e s everywhere seem to be i n v e r s e l y p r o p o r t i o n a l t o i s l a n d s i z e because {3) the r e l a t i v e e c o l o g i c a l d i s t i n c t n e s s i s g r e a t e r on s m a l l i s l a n d s than l a r g e ones". In many r e s p e c t s , the s m a l l l a k e s i n h a b i t e d by Gasterosteus c o n s t i t u t e an environment t h a t i s apparently l e s s s p a t i a l l y and t r o p h i c a l l y homogeneous than the i s l a n d s occupied by t h e l a c e r t a l i z a r d s . The f a c t t h a t Paxton and Enos l a k e s each c o n t a i n two e c o l o g i c a l l y and m o r p h o l o g i c a l l y d i s t i n c t s t i c k l e b a c k p o p u l a t i o n s supports the o b s e r v a t i o n of h a b i t a t h e t e r o g e n e i t y . T h i s phenomenum of c o - e x i s t i n g benthic and l i m n e t i c p o p u l a t i o n s i s a s s o c i a t e d with low numbers of pr e d a t o r s i n both l a k e s ( J . D. McPhail, p e r s . .comm.). T y p i c a l l y , s m a l l l a k e s i n h a b i t e d by G a s t e r o s t e u s i n the study r e g i o n a l s o are i n h a b i t e d by s t i c k l e b a c k predators and com p e t i t o r s . ., These i n c l u d e : t r o u t (Salmo c l a r k i and S. g a i r d n e r i ) , s c u l p i n s (Cottus spp.), and o f t e n salmon (On.cor hynchus s p p . ) , squawfish {P t y c ho c he i l us oreqonensis) and other s p e c i e s . In many s m a l l l a k e s , predation l i k e l y r e s t r i c t s the type of h a b i t a t Gasterosteus can e x p l o i t . Under such c o n d i t i o n s , s t i c k l e b a c k s tend t o l e a d a c r y p t i c , b e n t h i c a l l y - o r i e n t e d e x i s t e n c e . , The l i m n e t i c form found i n Paxton and Enos l a k e s o c c u p i e s , and feeds i n , the p e l a g i c zone. I t b u i l d s n e s t s i n exposed areas and p r o v i d e s an obvious t a r g e t f o r p r e d a t i o n . Indeed, the i n t r o d u c t i o n of coho salmon (0. k i s u t c h ) i n t o Paxton Lake l e d t o a d r a s t i c r e d u c t i o n i n numbers of l i m n e t i c s t i c k l e b a c k s (Larsen 1976, McPhail, pe r s . comm.). Thus, the a c t u a l h a b i t a t a v a i l a b l e f o r Gasterosteus i n most s m a l l l a k e s may be more narrowly de f i n e d than the p h y s i c a l and t r o p h i c p r o p e r t i e s of t h e l a k e s suggest. 95 The l o s s of g e n e t i c v a r i a b i l i t y through d i r e c t i o n a l s e l e c t i o n , much of i t undoubtedly a p p l i e d through p r e d a t i o n , i s a d i s t i n c t p o s s i b i l i t y i n these l a k e s , The Gasterosteus p o p u l a t i o n s of l a r g e l a k e s do n o t e x h i b i t the same r e d u c t i o n i n 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 r e l a t i v e t o marine p o p u l a t i o n s , S h i l e the degree of polymorphism i n l a r g e -lak e s t i c k l e b a c k s i s s l i g h t l y lower than i n marine p o p u l a t i o n s , the l e v e l of h e t e r o z y g o s i t y i s s l i g h t l y h i g h e r . These r e s u l t s correspond c l o s e l y w i t h those of Gorman e t a l * (1975) who found that p o p u l a t i o n s of L a c e r t a m e l i s e l l e n s i s on l a r g e i s l a n d s d i s p l a y e d high l e v e l s of h e t e r o z y g o s i t y . , They a t t r i b u t e d maintenance o f t h i s v a r i a b i l i t y t o the g r e a t e r e c o l o g i c a l v a r i e t y ( i . e . g r e a t e r niche width) on l a r g e i s l a n d s . , C e r t a i n l y , the l a r g e l a k e s of t h i s study p r o v i d e a much g r e a t e r range of h a b i t a t d i v e r s i t y than do the s m a l l l a k e s ; the s i z e of p e l a g i c r e g i o n s , e s p e c i a l l y , i n c r e a s e s d i s p r o p o r t i o n a t e l y with lake s u r f a c e a r e a . .However, l a r g e l a k e s possess the same range of p r e d a t o r s and c o m p e t i t o r s as s m a l l l a k e s , and to assume greater s t i c k l e b a c k e x p l o i t a t i o n of the p e l a g i c zone i n l a r g e l a k e s one must p o s t u l a t e reduced i n t e r s p e c i f i c i n t e r a c t i o n . Hanzer (1976) i n a d i e t a r y study of G a s t e r o s t e u s and j u v e n i l e sockeye salmon (0. nerka) i n Great C e n t r a l Lake found c o n s i d e r a b l e o v e r l a p i n food consumed by these two s p e c i e s , but concluded t h a t s e r i o u s c o m p e t i t i o n d i d not e x i s t . , Competition and p r e d a t i o n undoubtedly occur i n l a r g e l a k e s , but the e x t e n t depends t o a l a r g e degree on the amount of s p a t i a l and temporal s e g r e g a t i o n among s p e c i e s . The o p p o r t u n i t i e s f o r such s e g r e g a t i o n probably are g r e a t e r i n l a r g e 96 than i n small l a k e s . Thus, i t seems l i k e l y t h a t the absence of reduced 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 i n l a r g e l a k e s i s due, at l e a s t i n p a r t , to the range o f h a b i t a t s a v a i l a b l e . The morphological s i m i l a r i t y between the s t i c k l e b a c k s of l a r g e l a k e s and marine p o p u l a t i o n s (Hagen and G i l b e r t s o n 1972) may i n d i c a t e s i m i l a r i t y i n the s e l e c t i v e regimes i n the two types of h a b i t a t . Although, a c c o r d i n g to the time-divergence t h e o r y , t h i s m o r p h o l o g i c a l s i m i l a r i t y may not r e s u l t so much from s i m i l a r s e l e c t i v e regimes as from the absence of d i r e c t i o n a l s e l e c t i v e f o r c e s . The consequent l a c k of d i f f e r e n t i a t i o n , or slow e v o l u t i o n a r y r a t e , r e s u l t s i n the maintenance o f g e n e t i c v a r i a b i l i t y . , Since marine p o p u l a t i o n s are h i g h l y polymorphic, the o b s e r v a t i o n t h a t h e t e r o z y g o s i t y i s g r e a t e r i n l a r g e l a k e s than i n the ocean i n d i c a t e s t h a t the v a r i a n t a l l e l e s occur at higher f r e q u e n c i e s i n l a r g e l a k e s . Whether t h i s r e f l e c t s a r e d u c t i o n i n t h e s t r i n g e n t s e l e c t i o n a g a i n s t these a l l e l e s t h a t keeps them at u n i f o r m l y low f r e g u e n c i e s i n marine p o p u l a t i o n s , or r e s u l t s from a c t u a l s e l e c t i o n f o r the v a r i a n t s i n l a r g e l a k e s i s not c l e a r . Perhaps more s u r p r i s i n g than the high l e v e l s of h e t e r o z y g o s i t y i n l a r g e l a k e s are the even h i g h e r l e v e l s c h a r a c t e r i z i n g p o p u l a t i o n s i n h a b i t i n g l o w - l y i n g streams and swamps. These are much s m a l l e r p o p u l a t i o n s , occupying such h a b i t a t s as t r i b u t a r i e s t o the F r a s e r E i v e r , or s m a l l streams and ponds d r a i n i n g d i r e c t l y to the ocean as i n Sopke, or swamps adja c e n t to and o f t e n connecting the l a k e s of l a r g e watersheds. Temporal, r a t h e r than s p a t i a l , h e t e r o g e n e i t y of the environment i s most l i k e l y r e s p o n s i b l e f o r maintaining g e n e t i c v a r i a b i l i t y 97 i n these p o p u l a t i o n s . Hany of the c o a s t a l p o p u l a t i o n s occupy h a b i t a t s under t i d a l i n f l u e n c e . , O t h e r s , more d i s t a n t from the ocean, are s u b j e c t t o the f l o o d i n g t y p i c a l of lowland r e g i o n s . A l l a r e v u l n e r a b l e t o c l i m a t i c f l u c t u a t i o n * somewhat am e l i o r a t e d by lower e l e v a t i o n , as are the more ephemeral i s o l a t e d stream and swamp p o p u l a t i o n s . Despite both random and p r e d i c t a b l e h a b i t a t v a r i a b i l i t y over time, the g e n e r a l l y l a r g e r p o p u l a t i o n s i z e s i n these l o w - l y i n g streams (compared with those of i s o l a t e d stream l o c a l i t i e s ) undoubtedly reduce the e r o s i o n of g e n e t i c h e t e r o g e n e i t y through g e n e t i c d r i f t . another f a c t o r that may he l p maintain v a r i a b i l i t y i s gene flow. The F r a s e r S i v e r c o n s t i t u t e s a permanent d i s p e r s a l r o u t e f o r p o p u l a t i o n s occupying i t s t r i b u t a r i e s , and f l o o d i n g l i k e l y c r e a t e s t r a n s i e n t c o n n e c t i o n s among other l o w - l y i n g p o p u l a t i o n s . However, i f such gene flow does occur, i t i s i n s u f f i c i e n t to swamp the g e n e t i c d i f f e r e n t i a t i o n that i s apparent, even between many g e o g r a p h i c a l l y adjacent s i t e s . T h i s may r e f l e c t the st r e n g t h of the s e l e c t i v e f o r c e s a f f e c t i n g gene f r e q u e n c i e s i n these p o p u l a t i o n s . / Such f o r c e s , u n l i k e l y to be d i r e c t i o n a l , probably vary over time with environmental f l u c t u a t i o n . The present a n a l y s i s r e v e a l s f u r t h e r evidence f o r the i n f l u e n c e of n a t u r a l s e l e c t i o n on gene f r e q u e n c i e s . I f freshwater p o p u l a t i o n s are o f marine o r i g i n , and the founding genomes were t r u l y r e p r e s e n t a t i v e of marine gene f r e q u e n c i e s , then the average gene f r e q u e n c i e s of present day marine and freshwater p o p u l a t i o n s should be t h e same, u n l e s s they a re a l t e r e d by s e l e c t i o n . The data examined t o t h i s p o i n t a re compatible with the su g g e s t i o n of e v o l u t i o n of freshwater 98 p o p u l a t i o n s from marine s t i c k l e b a c k s through. i s o l a t i o n i n freshwater h a b i t a t s . / I f t h i s i s t h e case, l o c i monomorphic i n marine p o p u l a t i o n s should be monomorphic f o r the same a l l e l e i n freshwater p o p u l a t i o n s . L o c i polymorphic i n marine p o p u l a t i o n s may be polymorphic or monomorphic (through founder e f f e c t s ) i n freshwater ones. .,If- some freshwater p o p u l a t i o n s a re monomorphic at a l o c u s , then i n freshwater h a b i t a t s the f r e q u e n c i e s of f i x a t i o n f o r a l l e l e s at t h a t l o c u s s h o u l d be p r o p o r t i o n a l to t h e i r f r e q u e n c i e s i n marine p o p u l a t i o n s . As p r e d i c t e d , the two monomorphic l o c i , Ldh and Idh, are f i x e d f o r the same a l l e l e i n a l l p o p u l a t i o n s (marine and fre s h w a t e r ) ; however, a l l e l e f r e q u e n c i e s a t two polymorphic l o c i , Pgm and Mdh-3, d i f f e r s i g n i f i c a n t l y between the two environments. At the Pgm l o c u s , marine p o p u l a t i o n s a re c h a r a c t e r i z e d by high f r e g u e n c i e s of the most common a l l e l e , Pqm 1 0 0, lower f r e q u e n c i e s o f Pgm 9* and r e l a t i v e l y r a r e occurrences of other a l l e l e s . While P g m 1 0 0 i s a l s o the most common a l l e l e i n freshwater p o p u l a t i o n s , other a l l e l e s , Pgm 8 0, Pgm 9 0 and Pgm* 0 3, are o f t e n present at as h i g h , or higher, f r e g u e n c i e s . Pgm i s f i x e d (monomorphic) i n onl y two samples (Appendix V I I ) , both from fr e s h w a t e r , and i n both cases the lo c u s i s f i x e d f o r the common P g m x o ° a l l e l e . Ho s i g n i f i c a n t d i f f e r e n c e s i n a l l e l e f r e g u e n c i e s have been demonstrated amonq freshwater p o p u l a t i o n s occupying d i f f e r e n t h a b i t a t s . , At the Mdh-3 l o c u s , Mdh-3 5 5 predominates i n marine and Mdh-3100 i n freshwater p o p u l a t i o n s . Thus, average gene f r e q u e n c i e s d i f f e r between marine and freshwater h a b i t a t s . Moreover, while t h e average frequency of Mdh-3 5 S i s 0.853 and of Mdh-3*°° i s 99 0. 117 i n marine p o p u l a t i o n s , only t h r e e freshwater p o p u l a t i o n s a r e f i x e d f o r Mdh-3 S S and seven (excluding the i n t r o d u c e d Harion Lake population) are f i x e d f o r H d h - 3 1 0 0 (Appendix I I ) . , Although s e l e c t i o n i s i m p l i c a t e d as the f o r c e underlying the marine-freshwater a l l e l e frequency dichotomy, no s i g n i f i c a n t h e t e r o g e n e i t y i n Hdh-3 a l l e l e f r e q u e n c i e s i s apparent among freshwater p o p u l a t i o n s occupying d i f f e r e n t h a b i t a t s . This i n d i c a t e s t h a t the s e l e c t i o n r e s u l t i n g i n marine-freshwater Hdh-3 d i f f e r e n t i a t i o n i s a s s o c i a t e d with some fundamental d i f f e r e n c e between marine and freshwater e x i s t e n c e . Presumably, t h i s d i f f e r e n c e i s imposed e i t h e r e x t e r n a l l y by the environment or i n t e r n a l l y by p h y s i o l o g i c a l c o n s t r a i n t s . , Eaunich et a l . (1972) r e p o r t e d a s i m i l a r d i s t r i b u t i o n f o r a g e n e t i c a l l y c o n t r o l l e d hemoglobin v a r i a n t i n European p o p u l a t i o n s of G a s t e r o s t e u s . They examined r e s i d e n t freshwater p o p u l a t i o n s i n I t a l y and Germany and b r a c k i s h water p o p u l a t i o n s i n the c o a s t a l e s t u a r i e s and lagoons of the northwestern A d r i a t i c Sea. While freshwater p o p u l a t i o n s were monomorphic f o r the common hemoglobin A, c o a s t a l p o p u l a t i o n s e x h i b i t e d high f r e q u e n c i e s of hemoglobin B. Although o v e r a l l a l l e l e f r e q u e n c i e s a t the Ck l o c u s d i d not d i f f e r between marine and freshwater p o p u l a t i o n s , the frequency of Ck*°° was s i g n i f i c a n t l y higher i n p o p u l a t i o n s i n h a b i t i n g low-l y i n g streams and swamps than any other h a b i t a t , i n c l u d i n g the ocean. C k l ° ° was u n i v e r s a l l y present i n freshwater p o p u l a t i o n s occupying streams t h a t r e c e i v e s p r i n g breeding p o p u l a t i o n s of marine s t i c k l e b a c k s i n t h e i r lower reaches. Thus i t was present not o n l y i n the r e s i d e n t s t i c k l e b a c k s o f the L i t t l e Campbell (LC 100 18) , S a r i t a (SARIJ) and C h e h a l i s (Washington) (CHEHW) r i v e r s , but a l s o i n the l o w - p l a t e d ( l e i u r u s ) f i s h of mixed l e i u r u s -t r a c h u r u s samples c o l l e c t e d from the F r a s e r River (FRASR) ,. and from Lard (LAROC) and F u l l e r (FULCR) c r e e k s on Vancouver I s l a n d . Freshwater p o p u l a t i o n s i n t r i b u t a r i e s t o the F r a s e r (GIFFS and TEXAS) a l s o possessed the a l l e l e . I t was a l s o present i n r e s i d e n t p o p u l a t i o n s o f streams and r i v e r s from which marine s t i c k l e b a c k s were not sampled but l i k e l y e n t e r , such as the C o l g u i t z (CLQTZ), Serpentine (TZNEP) and Tugwell (SKOOS) s i t e s i n B.C., and the North Skagit (SKAGI) and Snohomish (EBEYI) s i t e s i n Washington. Although the l o w - p l a t e d p o p u l a t i o n s were i n some cases many k i l o m e t e r s upstream from r e g i o n s occupied by marine spawners, the presence of c k 1 0 0 seems d e f i n i t e l y a s s o c i a t e d with use of a stream by the two types of s t i c k l e b a c k p o p u l a t i o n s . I n c o n t r a s t , t h i s a l l e l e i s almost completely absent from lake p o p u l a t i o n s r e g a r d l e s s o f l a k e s i z e (exceptions a r e S a r i t a and Trout l a k e s ) . Small l a k e s t h a t possess two completely or p a r t i a l l y d i s t i n c t s t i c k l e b a c k p o p u l a t i o n s (Enos, Paxton, Goose, Cranby and Hiechhold) a l s o are e x c e p t i o n s . In Enos and Paxton, Ck*oo i s again present i n h i g h f r e g u e n c i e s i n the deep-bodied b e n t h i c s t i c k l e b a c k s and almost absent among the l i m n e t i c s . However, the b e n t h i c - l i k e s t i c k l e b a c k s of other s m a l l l a k e s , i n the absence of of the l i m n e t i c form, do not g e n e r a l l y possess the C k 1 0 0 a l l e l e (Appendix VI) . Thus, f o r Ck a l l e l e f r e g u e n c i e s , as f o r c e r t a i n morphological t r a i t s (Hagen and G i l b e r t s o n 1972), p o p u l a t i o n s of l o w - p l a t e d s t i c k l e b a c k s i n l a k e s d i s t a n t from the ocean are more s i m i l a r t o marine s t i c k l e b a c k s than are low-1 0 1 p l a t e d p o p u l a t i o n s i n h a b i t i n g streams i n which marine s t i c k l e b a c k s also, breed, although the mechanism u n d e r l y i n g t h i s apparent case of c h a r a c t e r displacement at the enzyme l e v e l i s unknown, the phenomenum appears widespread and may be c h a r a c t e r i s t i c of t r a c h u r u s - l e i u r u s i n t e r a c t i o n s . The h i g h l y s i g n i f i c a n t c o r r e l a t i o n s of C k 8 5 , P g m 1 0 3 and P g i - 2 1 0 0 f r e q u e n c i e s i n marine p o p u l a t i o n s with l o n g i t u d e r e s u l t from s m a l l , but c o n s i s t e n t , d i f f e r e n c e s i n a l l e l e f r e q u e n c i e s between marine p o p u l a t i o n s from the west c o a s t of Vancouver I s l a n d and those o f the more e a s t e r l y S t r a i t of Georgia. The frequency of the common C k 8 S a n d P g i - 2 1 0 0 a l l e l e s are lower, and of the r a r e Pgm 1 0 3 i s h i g h e r , i n the west c o a s t p o p u l a t i o n s {appendices V, VI and V I I ) . T h i s r e f l e c t s the s l i g h t l y g r e a t e r g e n e t i c h e t e r o g e n e i t y c h a r a c t e r i z i n g these p o p u l a t i o n s . , There a re s e v e r a l p o s s i b l e e x p l a n a t i o n s f o r t h i s g e n e t i c d i f f e r e n t i a t i o n : {1) s e l e c t i v e d i f f e r e n c e s between the r e g i o n s , {2) founder e f f e c t s ( i . e . d i f f e r e n t i a l p o s t g l a c i a l i n v a s i o n from northern and southern r e f u g i a ) or ( 3 ) g e n e t i c d r i f t , i f gene f l o w between the r e g i o n s i s low.„ Onderstanding of the a c t u a l mechanisms might be aided by the dete r m i n a t i o n o f a l l e l e f r e q u e n c i e s i n samples from more northern and southern extremes o f the tr a c h u r u s range. The s t r i k i n g e l e c t r o p h o r e t i c d i f f e r e n t i a t i o n between b e n t h i c and l i m n e t i c p o p u l a t i o n s w i t h i n Paxton and Enos l a k e s , coupled with the 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 between the two be n t h i c and two l i m n e t i c p o p u l a t i o n s , corresponds with morphological p a t t e r n s o f v a r i b i l i t y . . , I f e v o l u t i o n of the two forms occurred independently w i t h i n each of the l a k e s the 102 evidence f o r the s e l e c t i v e value of the e l e c t r o p h o r e t i c v a r i a n t s under such c o n d i t i o n s i s i m p r e s s i v e , e s p e c i a l l y as p r e l i m i n a r y evidence r e v e a l s no c o r r e l a t i o n between i n h e r i t a n c e of the m o r p h o l o g i c a l and 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 . {However, t h e p o s s i b i l i t y o f the l i n k a g e of m o r p h o l o g i c a l and e l e c t r o p h o r e t i c l o c i i s d e s e r v i n g of c l o s e r examination.). G e n e t i c a l l y d i s t i n c t 'species p a i r s * o f f i s h , m o r p h o l o g i c a l l y and e l e c t r o p h o r e t i c a l l y d i f f e r e n t i a t e d , occur i n a number of l a k e s . K i r k p a t r i c k and Selander (1979) s t u d i e d m o r p h o l o g i c a l l y d i s t i n c t sympatric p o p u l a t i o n s of w h i t e f i s h (Core q on us c l u pea form i s ) i n the A l l e g a s h B a s i n , Maine, l a k e s of the r e g i o n were populated by one or both of two w h i t e f i s h morphs, a normal and a dwarf. These forms d i f f e r e d not only i n growth r a t e but a l s o i n a number of m e r i s t i c t r a i t s . A l l e l e f r e q u e n c i e s a t s e v e r a l e l e c t r o p h o r e t i c l o c i i n d i c a t e d that the two forms w i t h i n a lake were g e n e t i c a l l y i s o l a t e d . Normal, or dwarf, p o p u l a t i o n s from d i f f e r e n t l a k e s were not, however, e l e c t r o p h o r e t i c a l l y more s i m i l a r than normal and dwarf p o p u l a t i o n s from the same or d i f f e r e n t l a k e s . K i r k p a t r i c k and Selander (1979) i n t e r p r e t e d the presence of s i m i l a r C. c l u p e a f o r m i s dwarf-normal s p e c i e s p a i r s i n a t l e a s t two other widely separated r e g i o n s of North America (Squanga Lake, Yukon T e r r i t o r y and Lake Opeongo, Ontario) as evidence o f s e v e r a l independent e v o l u t i o n s of the two " s p e c i e s " . S i m i l a r s t u d i e s on brown t r o u t ( A l l e n d o r f et a l . 1976) and A r c t i c char (Henricson and Nyman 1976, Nyman 1972, N i l s s o n and F i l i p s s o n 1971) i n Scandinavian l a k e s r e v e a l e d the presence of g e n e t i c a l l y d i s c r e t e s p e c i e s p a i r s . Henricson and Nyman (1,976) 1 03 compared gene f r e q u e n c i e s i n a l l o p a t r i c and sympatric p o p u l a t i o n s of two types o f a r c t i c char which occurred by themselves i n some l a k e s and c o - e x i s t e d (as a s p e c i e s p a i r ) i n other l a k e s . They found t h a t , i n sympatry, low r a t e s of gene flow between the two types had l e d to i n t r o g r e s s i o n at e l e c t r o p h o r e t i c l o c i , but as yet had not obscured t h e i r genetic d i s t i n c t n e s s . I t i s not c l e a r , f o r any of the s p e c i e s examined, whether the two c o - e x i s t i n g forms evolved i n sympatry o r during g e o g r a p h i c a l i s o l a t i o n . The p o s s i b i l i t y of the double i n v a s i o n of both Paxton and Enos l a k e s by common p o p u l a t i o n s of bent h i c and l i m n e t i c s t i c k l e b a c k s during p o s t g l a c i a l water l e v e l f l u c t u a t i o n s cannot be d i s p r o v e n . ,In <that case, the e l e c t r o p h o r e t i c a f f i n i t i e s of th e two be n t h i c and two l i m n e t i c p o p u l a t i o n s might simply be a r e f l e c t i o n of common ancestry r a t h e r than a response t o s i m i l a r s e l e c t i v e regimes. However, more support f o r the s e l e c t i v e theory i s provided by t h e g e n e t i c s i m i l a r i t y between these l a c u s t r i n e b e n t h i c and l i m n e t i c s t i c k l e b a c k s and t h e i r l o t i c c o u n t e r p a r t s , the L i t t l e Campbell l e i u r u s and trachurus p o p u l a t i o n s , a g a i n , e c o l o g i c a l and morphological s i m i l a r i t i e s correspond c l o s e l y t o 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 i n t h i s case the p o s s i b i l i t y of common o r i g i n of s i m i l a r phenotypes i s more remote. Hhatever t h e i r o r i g i n , a l l e l e f r e g u e n c i e s o f the ad u l t b e n t h i c and l i m n e t i c s t i c k l e b a c k s from Enos and Paxton i n d i c a t e t h a t w i t h i n each l a k e the two morphs c o n s t i t u t e g e n e t i c a l l y i s o l a t e d p o p u l a t i o n s . as i n the sympatric s p e c i e s of Scandinavian char, extremely low r a t e s of gene exchange may take 104 p l a c e . H y b r i d i z a t i o n between t h e two morphs occurs i n Paxton Lake ( J . D. M c P h a i l , pers. comm.) but few genomes among the a d u l t s surveyed were of p o s s i b l e h y b r i d o r i g i n , i n d i c a t i n g that h y b r i d s face a severe s e l e c t i o n d i f f e r e n t i a l i n t h e n a t u r a l environment. Lab-reared h y b r i d s d i s p l a y the expected h i g h l y heterozygous genotypes. Thus, l i k e the l e i u r u s and trachurus s t i c k l e b a c k s of the L i t t l e Campbell and other streams (Hagen 1967), b e n t h i c and l i m n e t i c s t i c k l e b a c k s act as good b i o l o g i c a l s p e c i e s . These r e s u l t s c o n t r a s t s h a r p l y with those of Avise (197 6) who examined a l l e l e f r e g u e n c i e s at e l e c t r o p h o r e t i c l o c i i n a G a s t e r o s t e u s p o p u l a t i o n dimorphic f o r p l a t e counts (high and low) i n the San Joaquin R i v e r , C a l i f o r n i a . Unlike the, l a c u s t r i n e b e n t h i c s and l i m n e t i c s o f the present study, these two types d i d not d i f f e r i n other morphological c h a r a c t e r i s t i c s such as body s i z e and shape.,Nor were e l e c t r o p h o r e t i c a l l e l e f r e g u e n c i e s a t polymorphic l o c i d i f f e r e n t between the high and low p l a t e d forms, and i n h e r i t a n c e of p l a t e phenotypes i n lab c r o s s e s supported the suggestion that the two types c o n s t i t u t e d a s i n g l e i n t e r b r e e d i n g p o p u l a t i o n . The f i n a l data p e r t a i n i n g to the i n f l u e n c e of s e l e c t i o n on gene f r e q u e n c i e s at t h e enzyme l e v e l i n G a s t e r o s t e u s i s provided by a comparison of a l l e l e f r e q u e n c i e s i n Chemainus and Marion l a k e s . Gene f r e q u e n c i e s were not determined i n Chemainus Lake p r i o r t o , nor i n e i t h e r l a k e immediately f o l l o w i n g , the 1974 i n t r o d u c t i o n of Chemainus s t i c k l e b a c k s i n t o Marion. However, a l l e l e f r e q u e n c i e s at a l l l o c i , i n c l u d i n g the polymorphic Pgm, Mdh-1 and Pgi-2, are not s i g n i f i c a n t l y heterogeneous among the 105 f o u r 1977 and 1978 Chemainus and 1977 and 1979 Marion samples. Any changes t h a t have taken p l a c e i n Chemainus Lake a t e l e c t r o p h o r e t i c l o c i s i n c e 1974 have occurred independently i n Marion Lake. On the other hand, morphological v a r i a b i l i t y has been monitored i n both l a k e s s i n c e 1974. , G r a d u a l , but s i g n i f i c a n t , change i n the degree of asymmetry o f , and mean» value of, p l a t e counts has o c c u r r e d i n Marion Lake, but not Chemainus (J.D. McPhail, pers. comm.) . T h i s i n d i c a t e s t h a t i f 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 at the l o c i examined i n t h i s study i s s u b j e c t t o s e l e c t i o n , i t responds t o d i f f e r e n t , or more slow l y t o the same, environmental v a r i a b l e s than do morphological t r a i t s . , Summary L e v e l s of h e t e r o z y g o s i t y and polymorphism i n 79 p o p u l a t i o n s °f G. a c u l e a t u s are comparable t o those c h a r a c t e r i s t i c of other v e r t e b r a t e s p e c i e s . In g e n e r a l , genotypic d i s t r i b u t i o n s conform wit h Hardy-Weinberg e x p e c t a t i o n s . , While gene f r e q u e n c i e s at polymorphic l o c i are s t a b l e over time w i t h i n p o p u l a t i o n s , they a r e h i g h l y heterogeneous among p o p u l a t i o n s . , Genetic d i s t a n c e s are much lower among marine than among freshwater s t i c k l e b a c k p o p u l a t i o n s . The d i s t a n c e s between marine and freshwater p o p u l a t i o n s are s i m i l a r t o those among freshwater l o c a l i t i e s . These f i n d i n g s are compatible with the suggestion t h a t freshwater ( l e i u r u s ) p o p u l a t i o n s i n southwestern fi. C. are polyph y l e t i c , and have descended from marine (trachurus) s t i c k l e b a c k s i s o l a t e d i n freshwater h a b i t a t s during p o s t g l a c i a l f l u c t u a t i o n s i n water l e v e l s . A l t e r n a t i v e l y , these r e s u l t s can 106 be e x p l a i n e d by p o s t u l a t i n g d i f f e r e n c e s i n s e l e c t i v e f o r c e s among freshwater, and between marine and f r e s h w a t e r , environments. Comparisons of g e n e t i c v a r i a b i l i t y w i t h i n and between the Bear and Somass r i v e r watersheds do not provide s t r o n g support f o r e i t h e r h y p o t h e s i s . , Although t r a c h u r u s p o p u l a t i o n s are g e n e t i c a l l y more homogeneous than l e i u r u s p o p u l a t i o n s , a c l e a r geographic d i s t i n c t i o n i n a l l e l e f r e g u e n c i e s at the Pgm, Pgi-2 and Ck l o c i s e p a r ates marine s t i c k l e b a c k s i n h a b i t i n g wafers o f f the west c o a s t of Vancouver I s l a n d from those occupying the S t r a i t of Georgia. No c o n s i s t e n t geographic p a t t e r n s i n a l l e l e f r e q u e n c i e s are apparent among the h i g h l y heterogeneous freshwater p o p u l a t i o n s . Gasterosteus p o p u l a t i o n s i n h a b i t i n g the ocean, l a r g e l a k e s and l o w - l y i n g streams a r e more polymorphic and heterozygous than those occupying s m a l l lakes and i s o l a t e d streams. . Both s t o c h a s t i c (founder e f f e c t s and g e n e t i c d r i f t ) and d e t e r m i n i s t i c ( n a t u r a l s e l e c t i o n ) f a c t o r s can be invoked t o account f o r these p a t t e r n s . T h e i r r e l a t i v e c o n t r i b u t i o n s t o present l e v e l s of v a r i a b i l i t y remain s p e c u l a t i v e . , Evidence f o r the e f f e c t o f s e l e c t i o n on a l l e l e f r e g u e n c i e s i n c l u d e s d i f f e r e n c e s i n a l l e l e d i s t r i b u t i o n s a t the Pgm and Mdh-3 l o c i between marine and freshwater p o p u l a t i o n s , and e l e v a t e d f r e q u e n c i e s of C k 1 0 0 i n the l e i u r u s p o p u l a t i o n s of l o w - l y i n q streams. The s i m i l a r i t y o f e l e c t r o p h o r e t i c and morphological a f f i n i t i e s among the ben t h i c and l i m n e t i c 'species p a i r s * i n Paxton and Enos l a k e s , and the l e i u r u s and trachurus s t i c k l e b a c k s of the L i t t l e Campbell R i v e r , i s a l s o s u g g e s t i v e of 107 a s e l e c t i v e i n f l u e n c 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 . However, morphological divergence between the s t i c k l e b a c k s of Chemainus and Marion l a k e s i s as yet unaccompanied by a l l e l e freguency d i f f e r e n t i a t i o n , i n d i c a t i n g that 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 may be l e s s r e s p o n s i v e t o environmental change than i s morphology. 108 LITERATURE CITED A l l e n d o r f , F. , H., N. Ryman, A. Stenaek and G. S t a h l . 1976.. Genetic v a r i a t i o n i n Scandinavian p o p u l a t i o n s of brown t r o u t (Salmo t r u t t a L . ) : evidence o f g e n e t i c a l l y d i s t i n c t sympatric p o p u l a t i o n s . H e r e d i t a s 82: 19-24., A l l e n d o r f , F. 8., F. H. . O t t e r and B., P., May. „1975. Gene d u p l i c a t i o n w i t h i n the f a m i l y Salmon!dae. I I . D e t e c t i o n and deter m i n a t i o n of the g e n e t i c c o n t r o l of d u p l i c a t e l o c i through i n h e r i t a n c e s t u d i e s and the examination of p o p u l a t i o n s . In: Isoenzymes IV. G e n e t i c s and E v o l u t i o n (C. L. Harkert, ed.) , pp. 415-432. A l l e n d o r f , F. „•». and F. H. O t t e r . 1979. P o p u l a t i o n g e n e t i c s . I n : F i s h P h y s i o l o g y V o l . VII. B i o e n e r g e t i c s and Growth (W. S. Hoar, D. J . Randall and J . R. B r e t t , eds.), pp. 407-454. Academic Press. , Avise, J . C. 1976. 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E v o l u t i o n 27: 593-600. S t e e l , R. G. D. and J . H. , T o r r i e . 1960. , P r i n c i p l e s and Procedures i n S t a t i s t i c s . McGraw-Hill, Hew York.,481 pp. Tanaka, A. , A. Fukunaga and K. O i s h i . , 1976. .Studies on the sex-s p e c i f i c l e t h a l s of D r o s o p h i l a melanogaster. .. I I . F u r t h e r s t u d i e s on a m a l e - s p e c i f i c l e t h a l gene, m a l e l e s s . Genetics 84: 257-266. , Thome, C. , J. R. , L. . I. Grossman and H. ,o» .Kaplan. , 1963. S t a r c h -g e l e l e c t r o p h o r e s i s of malate dehydrogenases. , Biochim. Biophys. Acta 73: 193-2 03. Tomaszewski, E. „ K., B. . E. , S c h a f f e r and F. M. Johnson. 1973. Isozyme genotype-environment a s s o c i a t i o n s i n n a t u r a l p o p u l a t i o n s of the h a r v e s t e r ant, Pogonomyrmex badius., G e n e t i c s 75: 405-421., Turner, B, M., R, A., F i s h e r and H. H a r r i s . , 1975. Post-t r a n s l a t i o n a l a l t e r a t i o n s of human e r y t h r o c y t e enzymes. I n : Isozymes I . , Molecular S t r u c t u r e (C. L. Markert, ed.), pp. 781-795.,Academic press. U t t e r , F. M. and H. 0. Hodgins. 1970. Phosphoglucomutase polymorphism i n sockeye salmon. Comp. Biochem. P h y s i o l . 36: 195-199. Ward, R. H.,and C. F. S i n g . 1970. A c o n s i d e r a t i o n of the power of the c h i - s q u a r e t e s t t o d e t e c t i n b r e e d i n g e f f e c t s i n n a t u r a l p o p u l a t i o n s . Amer. Natur. ,104: 355-365. Wheat, T. E. and G. S. Whitt. 1971. In vivo and i n v i t r o molecular h y b r i d i z a t i o n of malate dehydrogenase isozymes. 120 E x p e r i e n t i a 27: 647-648., Whitt, 6. / S. 1970. Genetic v a r i a t i o n of supernatant and m i t o c h o n d r i a l malate dehydrogenase isozymes i n the t e l e o s t Fundulus h e t e r o c l i t u s . E x p e r i e n t i a 26: 734-736. Wcotton, fi., J . 1976. The B i o l o g y of the S t i c k l e b a c k s . Academic P r e s s , London. 387 pp. Workman, P., L. . 1969. The a n a l y s i s of simple genetic polymorphisms. Human B i o l . 41: 97-114. 121 APPENDIX I L o c a t i o n s and dates of G. a c u l e a t a s sample c o l l e c t i o n s . 1. Marine P o p u l a t i o n s Englishman R i v e r , e s t u a r y , Vancouver I s l a n d (ENG1R). , June 1977. 49°20« 124<M7» Horseshoe Bay, west of Vancouver (HORSB) . Nov. 1977. 49°23* 123°16« Metchosin Lagoon, Sooke, Vancouver I s l a n d (WITLG) . June 1977. 48°23» 123°31« L i t t l e Campbell R i v e r , estuary, White Rock, B.C. (LC 19). June 1978. 49°01» 122°46* Cowichan Bay, Vancouver I s l a n d (COWIB). June 1977. 48°44» 123°38» Pender Harbour, Madeira Park Wharf, S e c h e l t Peninsula (MDERA). A p r i l 1978. 49°37« 124°01» Sooke R i v e r , Vancouver I s l a n d (SOOKP) . June 1977. 48°24» 123°42« K o k s i l a h River, e s t u a r y , Vancouver I s l a n d (KOKSR). June 1977. 48°46» 123°40« Stream e n t e r i n g O c l u e l e t I n l e t , 3 km N of Thornton Creek, Vancouver I s l a n d (SLMNC) . J u l y 1978. 48°59« 125°34« Grappler I n l e t , Vancouver I s l a n d (GRAPI). May 1978. 48°50 f 125<>07» Small stream near Bamfield, Vancouver I s l a n d (BAMFS). Aug. 1978. 48°52* 125°06« S a r i t a R i v e r , e s t u a r y , Vancouver I s l a n d (SARIE)., May 1978. 48°54» 125°01' Haines I s l a n d , lagoon, W of Vancouver I s l a n d (HAINL). May 1978. 48°50« 125°12» Congreve I s l a n d , 8 of Vancouver I s l a n d (CONGR) . „ May 1978. 48°56* 125°02« Santa Maria I s l a n d , W of Vancouver I s l a n d (SANMA). May 1978. 480 53' 125°01« C h e h a l i s R i v e r , at Wensall Ed., Wash. S t a t e (CHEHW) . June 1978. 46°59* 123°22» 2. Large Lake P o p u l a t i o n s Cowichan Lake, Vancouver I s l a n d (COWIL) . , Sept. 1977. 48°50* 124°12» S a r i t a Lake, Vancouver I s l a n d (SARIL). , May 1978. 48°55« 124°52» Sakinaw Lake, S e c h e l t P e n i n s u l a (SARIN). May 1978. 49°39« 124°03* Sproat Lake, Vancouver I s l a n d (SPROT) . , May and Sept. 1978. 49°18» 124°56* McCreight Lake, Vancouver I s l a n d (MCRTL). June and Sept. 1978. 50°17» 125°39« Great C e n t r a l Lake, Vancouver I s l a n d (GRCEN). May 1978. 49°19« 124°59» S t e l l a Lake, Vancouver I s l a n d (STELL) . June 1978. 50°17» 125°30« Roberts Lake, Vancouver I s l a n d (ROBTL). June 1978., 50O14* 125°32« 122 3. Small Lake P o p u l a t i o n s Chemainus Lake, Vancouver I s l a n d (CHEML). Sept. 1977 and Oct. 1978. 48°55* 123°45' F u l l e r Lake, Vancouver I s l a n d (FOLLL). Sept. 1977., 48°54» 123°43» Jacobs (Marion) Lake, Haney, B.C. (MARNL). Aug. , 1977 and June 1979. 49°19» 122°33* Lake Erro c h (Sguakum Lake), B.C. (LKERB) . Aug. 1978. 49°14* 122°0 1« H o t e l Lake, S e c h e l t P e n i n s u l a (HOTEL):. A p r i l 1978. 49°38» 124°03* K l e i n Lake, S e c h e l t P e n i n s u l a (KLEIN) . May 1978. ,49°44« 124°03' Pag Lake, S e c h e l t Peninsula (PAfiLK). A p r i l 1978. 49°37* 124°02' Trout Lake, Sec h e l t P e n i n s u l a (TBOOT). A p r i l 1978. 49°31« 123°53* Garden Bay Lake, S e c h e l t Peninsula (GABBY). Hay 1978. 49°38* 124°02« Blackwater Lake, Vancouver I s l a n d (BLACK). June and Sept. 1978. 50°11« 125°35« P a t t e r s o n Lake, Vancouver I s l a n d (PATEB) . Hay and Sept. 1978 49°21* 125°00* D e v i l ' s Den Lake, Vancouver I s l a n d (DEVIL). Hay 1978. 49°15* 124°52« Sumner Lake, Vancouver I s l a n d (SOHNL)., Hay and Sept. 1978., 49°22» 124°59« Hud Lake, Vancouver I s l a n d (HODLK). June and Sept. 1978. ,50° 1 2* 125°33» Lovry Lake, Vancouver I s l a n d (LOHBL). May and Sept..1978. 49°24« 125°08* McCoy Lake, Vancouver I s l a n d (MCOYL). May and Sept. , 1978. . 49°16* 124<>53« C e c i l Lake, Vancouver I s l a n d (CECIL). June and Sept. 1978. 50°14* 125033* Morgan Lake, Vancouver I s l a n d (MOBGL). June and Sept. 1978. 50° 13* 125°33» Ormond Lake, Vancouver I s l a n d (QBMND) . June 1978.,50°11* 125°31* F a r e w e l l Lake, Vancouver I s l a n d (FABBL). June and Sept. 1978. 50O12* 125035* Cedar Lake, Vancouver I s l a n d (CEDAR) . June and Sept. 1978. 50o12* 125034* Paxton Lake, Texada I s l a n d (PAXTB and PAXTL). Oct. 1977. 49°43* 124°31* Enos Lake, Vancouver I s l a n d (ENOSB and ENOSL).. Oct. 1977 and Sept. 1978. ,49017* 124°09* Goose Lake, Haney B.C. (GOOSE). Nov. 1977. 49°18* 122°36« Hiechhold Lake, Texada I s l a n d (HECHL) . Oct. 1977. 49°46» 124°35* Cranby Lake, Texada I s l a n d (CBANL) . Oct. 1977. 49°42* 124 o30» 4. Low-lying Stream and Swamp P o p u l a t i o n s Tugwell Creek, Sooke, Vancouver I s l a n d (SKOOS) . , June 1977. 48°22* 123051* Slough N of W i l f r e d Creek, Ship P e n i n s u l a , Vancouver I s l a n d (SHIPC). June 1977. 49°30« 124°48* L i t t l e Campbell B i v e r , 15 km upstream from mouth. White Bock, 123 B.C. . (LC 18) . March and June 1978. ,49°02* 122°39* G i f f o r d Slough, McLennan Creek, F r a s e r R i v e r V a l l e y JGIFFS). May 1977. 49<>07 • 122°20» Yorkson Creek, F r a s e r River V a l l e y (TEXAS).,May 1977. 49°12* 122°39* Stream e n t e r i n g Deep Cove, Saanich I n l e t , Vancouver I s l a n d (PATBS). June 1977.,48°39» 123°27« Snohomish R i v e r , slough near Ebey I s l a n d , Wash. S t a t e (EBEYI). . June 1978. 48°01* 122°09* North S k a g i t R i v e r , Wash. S t a t e (SKAGI) . June 1978. 48°21 * 122°27* Slough on Westham I s l a n d , S arm F r a s e r R i v e r e s t u a r y (MOOSE). May 1978. 49°06' 123° 10* Roadside swamp 3 km NE of McCreight Lake, Vancouver I s l a n d (SMODL). June 1978. ,50°19* 125°36» Nathan Creek, F r a s e r River V a l l e y (NATHN) • May 1977. 49°08* 122°28* C o l g u i t z R i v e r , Vancouver I s l a n d (CLQTZ) . Sept. 1977. 48°28* 123°24* Roadside pond 2 km S of McCreight Lake, Vancouver I s l a n d (MCRPD) . June 1978.,50°16* 125°39« S a r i t a R i v e r , j u n c t i o n o f N and S arms, Vancouver I s l a n d (SARIJ). 48°54» 124°59« Serpentine R i v e r , pond near Bothwell Park, Surrey, B.C. (TYNEP). March, June and Aug. 1978. June 1979. 49° 10» 122°45» C h e h a l i s R i v e r , a t Hoquiam Beach Road, Wash. St a t e (HOQUB) . Oct. 1978. 47°00« 123°53« Roadside slough 2 km E of O t t e r Point, Sooke, Vancouver I s l a n d (OTTER). June 1977. 48°22« 123°48* Swamp between Blackwater and F a r e w e l l l a k e s , Vancouver I s l a n d (FARBL) . June and Sept. ,1978. 50° 11 • 125°35« 5. . I s o l a t e d Stream Po p u l a t i o n s Keogh R i v e r , 30 km upstream from mouth, Vancouver I s l a n d (KEOGH) . „ May 1977. . 50032* 127°13« Roadside swamp N of F a r e w e l l Lake, Vancouver I s l a n d (FARSW). June 1978. 50°13' 125°35' Roadside pond NW of T a y l o r Arm, Sproat Lake, Vancouver I s l a n d (SPRPD). May and Sept. 1978. 49°17* 125 014« S a l z e r River, near C e n t r a l i a , Wash. S t a t e (SLZER). June 1978. 46<>42» 122057* Dry Run Creek, C h e h a l i s River drainage. Wash.,State (DRYEN), June 1978. 47°08* 123°20* 6. Mixed P o p u l a t i o n s F r a s e r R i v e r , S arm near Woodward I s l a n d (FRASR). May 197 8. 49°07* 123°10« Lard Creek, Vancouver I s l a n d (LABDC) • , June 1978. 49°40 • 124°58» F u l l e r Creek, Vancouver I s l a n d (FOLCR) . June 1977. 48°55» 123°42* L i t t l e Campbell R i v e r , 2.5 km upstream from mouth, White Rock, B.C. (LC 40). June 1978. 49°01* 122°45» 124 APPENDIX I I A l l e l e frequency d i s t r i b u t i o n at the Mdh-3 l o c u s . Sample s i z e s are the number of f i s h (one-half the number of genes) examined. Expected numbers of heterozygotes (in brackets) were c a l c u l a t e d f o r Hardy-Weinberg c o n d i t i o n s . Population Sample S i z e A l l e l e Frequencies Mdh-3ioo Mdh-3ss Heterozygotes Observed (Exp) Hetero-z y g o s i t Marine ENGLR 30 0.000 1.000 0 (0) 0.000 HOB SB — — — — WITLG 24 0.125 0.875 2 (.5) 0.219 LC 19 57 0.430 0.570 31 (28) 0.490 COW IB 27 0.056 0.944 3 (3) 0. 106 HDEBA 30 0. 117 0. 883 7 (6) 0. 207 SOOKP 31 0. 097 0.90 3 4 (5) 0. 175 KOKSB 69 0.188 0.812 20 (21) 0.305 SLMNC 53 0. 160 0. 840 11 (14) 0.269 GBAPI 41 0.183 0.817 13 (12) 0. 299 BAMFS 34 0. 176 0.824 6 (10) 0.290 SAB IE 44 0.159 0.84 1 14 (12) 0.267 HAINL 23 0.217 0.783 4 (8) 0. 340 CONGB 42 0.095 0.905 6 (7) 0. 172 SANMA 49 0.112 0.888 9 (10) 0. 199 CHEHW 16 O.094 0.906 3 (3) 0.170 Large Lake COWIL 37 0.000 1.000 0 (Q) 0.000 SAB XL 57 0. 816 0. 184 9 (17)* 0.300 SAKIN 11 0.364 0.636 8 (5) 0. 463 SPBOT 137 0.588 0. 412 59 (66) 0.485 MCRTL 258 0.764 0.236 96 (9 3) 0. 361 GBCEN 53 0. 46 2 0.538 21 (26) 0.497 ST ELL 19 0.895 0. 105 4 (<•) 0.188 BOBTL 21 0.524 0.476 12 (10) 0.499 Small Lake CHEML 83 1.000 0.000 0 (0) 0.000 FU1LL 32 0.641 0.359 15 (15) 0.460 MAHNL 75 1.000 0.000 0 (0) 0.000 LKEBE 46 0.359 0.641 27 (21) 0. 460 HOTEL 114 0.570 0. 430 52 (56) 0.490 KLEIN 45 1.000 0.000 0 (0) 0.000 PAQLK 41 0.976 0.024 2 (2) 0.047 TBOUT 50 0.840 0. 160 16 (13) 0.269. GABBY 14 0.964 0.036 1 (1) 0.069 BLACK 250 0.500 0.500 118(125) 0.500 PAT EE 106 0.962 0.038 8 (8) 0.07 3 DEVIL 84 0. 464 0. 536 46 (42) 0.497 SOMNL 66 0.992 0.008 1 (1) 0.016 HODLK 59 0.000 1.000 0 (0) 0.000 125 LOW EL 47 0.298 0.702 18 (20) 0. 418 MCOYL 129 0.926 0.074 15 (18) 0. 137 CECIL 103 0.267 0.733 45 (40) 0.391 MORGL 82 0.000 1.000 0 (0) 0.000 GBMND 62 0.040 0.960 5 (5) 0.077 FARWL 139 0.500 0.500 79 (70) 0.500 CEDAE 108 0. 213 0.787 38 (36) 0.335 PAXTB 112 0.987 0.013 3 (3) O.026 PAXTL 41 0.707 0.293 18 (17) 0.414 ENOSB 124 1.000 0.000 0 (0) 0.000 ENOSL 152 0.822 0. 178 38 (44) 0.293 GOOSE 63 0.603 0.397 34 (30) 0.479 HECHL 28 0.643 0. 357 10 (13) 0.459 CRANL 48 1.000 0.000 0 (0) 0.000 Low-1ying Stream or Swamp SKOOS — — — — — — SHI PC 27 0. 130 0.870 5 (6) 0.226 LC 18 87 0.805 0.195 32 (27) 0.314 GIFFS 13 0.923 0. 077 2 (2) 0. 142 TEXAS 19 0.684 0.316 12 (8) 0.432 PATBS 49 1.000 0.000 0 (0) 0.000 EBEYI 68 0. 507 0.493 25 (34)* 0.500 SKAGI 37 0.541 0.459 16 (18) 0.497 MOOSE 36 0.347 0.653 15 (16) 0.453 SMODL 78 1.000 o.ooo 0 (0) 0.000 NATHH 39 0.295 O.705 17 (16) 0.416 CLQTZ 47 0.766 0.234 16 (17) 0.358 MCRPD 98 0.786 0.214 38 (33) 0.336 SAEIJ 39 0.60 3 0.397 13 (19) 0.479 TYNEP 322 0. |38 0.862 53 (77) ** 0.238 HOQOB 33 0.030 0.970 0 (2) 0.058 GTTER 117 0. 295 0.705 49 (49) 0.416 FABBL 172 0.506 0.494 84 (86) 0.500 I s o l a t e d Stream or Swamp KEOGH 45 1.000 0.000 0 (0) 0.000 FARSW 74 0.372 0.628 25 (35)* 0.467 SPBPD 166 0.726 0.274 59 (66) 0.398 SLZER 61 0.189 0.811 13 (19) 0.307 DRYRN 50 0.530 0. 470 23 (25) 0.498 M ixed FRASR 100 0. 390 0.610 46 (48) 0.47 6 LARDC 85 0.729 0. 271 36 (34) 0.395 FOLCR 63 0.230 0.770 19 (22) 0. 354 LC 40 43 0.419 0.581 16 (21) 0.487 * P < 0.05 ** P < 0.01 126 APPENDIX I I I A l l e l e frequency d i s t r i b u t i o n a t the Mdh-1 l o c u s . Sample s i z e s are the number o f f i s h {one-half the number of genes) examined. Expected numbers of heterozygotes ( i n brackets) were c a l c u l a t e d f o r Hardy-Beinberg c o n d i t i o n s . P o p u l a t i o n Sample A l l e l e F r e g u e n c i e s Heterozygotes Hetero-S i z e Mdh-1* oo Mdh -1 8 2 Other Observed (Exp) z y g o s i t y Marine ENGLB 29 0.914 0.086 5 (5) 0.157 HOESB 30 0.967 0,033 2 (2) 0.064 HITLG 24 1.000 0.000 0 (0) 0,000 LC 19 57 0.974 0.026 . 3 (3) 0.051 COHIB 27 1.000 0.000 0 (0) 0.000 HDEBA 16 0.969 0.031 1 (1) 0.060 SOOKP 32 0.922 0.078 5 (5) 0.144 KOKSB 65 0.977 0.023 3 (3) 0.045 SLMNC 53 0.991 0.009 1 (1) 0,0 18 GBAPI 41 0.988 0.0 12 . 1 (1) 0.024 BAMFS 34 0.912 0.088 6 (5) 0.161 SABIE 44 0.909 0.091 6 (7) 0. 165 HAINL 23 1.000 0.000 0 (0) 0.000 CONGE 36 0.944 0,056 4 (4) 0,106 SANMA 44 0.943 0. 057 5 (5) 0.108 CHEH8 16 0.844 0.156 — — — 5 (4) 0.263 Large Lake CORIL 37 0.946 0.054 - - - 4 (*»)• 0.102 SABIL 57 0.658 0.342 29 (26) 0.450 SAKIN 11 0. 818 0. 182 4 (3) 0.298 SPEOT 138 0.862 0.138 . 30 C33) 0.238 MCBTL 260 0.856 0. 1 44 61 (64) 0.247 GBCEN 53 0.981 0.019 2 (2) 0,037 STELL 19 0.842 0. 158 6 (5) 0.266 BOBTL 21 1.000 o.ooo — — — 0 (0) 0.000 Small Lake CHEML 81 0.975 0.025 — - 4 H) 0.049 FOLLL 37 0.73 0 0.270 16 (15) 0.394 MABNL 75 0.933 0.067 10 (9) 0.125 LKEBB 46 0.967 0.033 : 3 (3) 0.064 HOTEL 114 0.987 0.013 . 3 (3) 0.026 KLEI N 45 0.711 0.289 20 (18) 0.411 PAQLK 41 1.000 0.000 0 (0) 0.000 TBOUT 50 1.000 0.000 0 (0) 0.000 GABBY 14 1.000 0. 000 •- 0 (Q) 0.000 BLACK 256 0.984 0.016 8 (8) 0,0 31 PAT EE 107 1.000 0.000 0 (Q) 0.000 DEVIL 85 1.000 0.000 0 (0) 0.000 SUMNL 66 0.992 0.008 1 (1) 0.0 16 MODLK 59 1.000 0.000 0 (Q) 0.000 127 LOW EL 47 0. 989 MCOYL 130 0. 988 CECIL 103 1. 000 MORGL 82 1. 000 ORHND 62 1. 000 FAB WL 139 0. 950 CEDAB 108 1. 000 PAXTB 113 0. 965 PAXTL 44 0. 977 ENOS B 125 1. 000 ENOSL 154 1, 000 GOOSE 66 1» 000 HECHL 37 1. 000 CRANL 48 1. 000 Low-1 ying Stream or S w amp SKOOS 35 0. 957 SHI PC 27 1* 000 LC 18 87 0. 983 6IFFS 13 0. 923 TEXAS 26 0. 942 PATBS 49 0. 510 EBEYI 68 0. 831 SKA GI 37 0. 946 MOOSE 36 0. 903 SMODL 78 0. 583 NATHN 41 0. 854 CLQTZ 47 1. 000 MCRPD 100 0. 875 SARIJ 39 0. 897 TYNEP 323 1. 000 HOQOB 33 0. 985 OTTER 117 0. 996 FARBL 172 0. 953 I s o l a t e d Stream o r Swamp KEOGH 45 1. 000 FARSW 75 0. 920 SPRPD 166 0. 976 SLZER 61 0. 402 DRYRN 50 1. 000 Mi xed FRASR 106 0, 976 LARDC 88 0. 983 FULCR 63 0. 968 LC 40 45 0. ,967 0.011 1 (D 0.022 0.0 12 3 (3) 0.024 0.000 0 (0) 0.000 0.000 0 (0) 0.000 0.000 0 (Q) 0.000 0.050 14 (13) 0.095 0.000 • 0 (0) 0.000 0.035 6 (8) 0.068 0.023 2 {2) 0.045 0.000 « — 0 (0) 0.000 0.000 0 (0) 0.000 0.000 0 (0) G.000 0.000 0 (0) 0.000 0.000 — •—• — 0 (Q) 0.000 0.043 _>»* 3 (3) 0.082 0.000 --- 0 (0) 0.0 00 0.017 — 3 (3) 0.033 0.077 2 (2) 0.142 0. 058 — . • 3 (3) 0.109 0.4 90 — 20 (24) 0.500 0.154 0.015 19 (19) 0.286 0.054 4 (4) 0.102 0.803 0.014 5 (6) 0. 178 0.417 — — • 39 (38) 0.4 86 0.146 k 8 (1.0) 0.249 o.ooo 0 (0) 0.000 0. 125 19 (22) 0.219 0.103 8 (7) 0.185 0.0 00 0 CP) 0.000 0.015 1 (!) 0.030 0.004 1 (D 0.008 0.047 16 (15) 0.090 0.000 0 (0) 0.000 0.080 12 (11) 0.147 0.024 8 (8) 0.047 0.000 0.598 25 (29) 0.481 0.000 — — —• 0 (0) 0.000 0.024 5 (5) 0.047 0.017 3 (3) 0.033 0.0 32 . 4 (4) ! 0.062 0.033 — - 3 (3) 0.064 128 APPENDIX IV A l l e l e frequency d i s t r i b u t i o n at the Pgi-1 l o c u s . Sample s i z e s are the number of f i s h (one-half the number o f genes) examined. Expected numbers o f heterozygotes ( i n brackets) were c a l c u l a t e d f o r Hardy-Weinberg c o n d i t i o n s . Population Sample A l l e l e Freguencies Heterozygotes Hetero-S i z e P g i - 1 1 0 0 P g i - 1 4 0 S Other observed(Exp) z y g o s i t y M a r i n e ENGLB 30 0.950 0. 033 0.017 1 13) 0,096 H0BSB 37 1.000 0.000 0 (0) 0.000 WITLG 28 0.964 0.036 0 (2) 0.069 LC 19 57 1,000 0.000 0 (0) 0.000 COW IB 27 1.000 0.000 — 0 (0) 0.000 HDEBA 18 1.000 0.000 0 CP-) 0.000 SGOKP 33 0.985 0.015 1 (1) 0.030 KOKSB 64 1.000 0.000 0 (Q) 0.000 SLHNC 53 0.962 0.038 0 0,073 GBAPI 41 0,976 0.024 — . 0 (2) 0*047 BAHFS 34 0.971 0.029 0 (2) 0.056 SABIE 44 1.000 0.000 0 (P) 0.000 HAINL 23 1.000 0.000 0 (0) O.OOO CONGB 35 0.971 0.029 . 0 (2) 0.056 SANHA 30 1.000 0.000 — - 0 (Q) 0.000 CHEHW 16 1.000 0.000 ——— 0 (Pi o.ooo Large Lake COWIL 37 0. 973 0.027 ——- 0 (2) 0.053 SABIL 57 1.000 0.000 — — 0 (0) 0,000 SAKIN 11 0.955 0.045 1 (D 0.086 SP80T 138 1. 000 0.000 _,— 0 (0) 0.000 MCBTL 218 1.000 0.000 0 (Q) 0.000 GBCEN 53 0.821 0. 179 11 (16) 0.2 94 STELL 18 0.972 0.000 0.028 1 in 0.054 BOBTL 21 1.000 0.000 ——— 0 (Q) 0.000 Small Lake CHEHL 79 1.000 0.000 0 (0) 0.000 FDLLL 37 i.ooo 0.000 0 (0) 0.000 HABNL 75 1.000 0.000 0 (Q) 0.000 LKEBB 46 1.000 0.000 0 (Q) o.ooo HOTEL 114 0.996 0.000 0.004 1 (1) 0.008 KLEIN 45 0.978 0.022 0 (2) 0.0 43 PAQLK 41 1,000 o.ooo 0 (0) O.OOO TBODT 50 1.000 0.000 0 (0) 0.000 GABBY 14 1.000 0.000 0 (0) 0.000 BLACK 170 0.994 0.003 0.003 2 (2) 0.012 PATEB 107 0.991 0.009 0 (2) 0.018 DEVIL 85 1. 000 0.000 0 (0) 0.000 SOHNL 66 1.000 0.000 -• 0 (0) 0.000 HODLK 59 1.000 o.ooo 0 (0) 0.000 129 IOW EL 30 1. 000 MCOYL 129 1 . 000 CECIL 103 1 . 000 MORGL 81 1. 000 ORMND 62 1. 000 FARWL 139 1. 000 CEDAR 107 0. 991 PAXTB 113 1. 000 PAXTL 44 O . 977 ENOSB 125 1. 000 ENOSL 154 1. 000 GOOSE 67 1. 000 HECHL 37 1. 000 CRANL 48 1. 000 Low-lying Stream o r S wamp SKOOS 35 1. 000 SHI PC 27 1. 000 LC 18 50 1. 000 GIFFS 13 1. 000 TEXAS 26 1. 000 PATBS 49 1. 000 EBEYI 68 0. 993 SKAGI 37 0. 865 MOOSE 36 0. 986 SMDDL 78 1. 000 NATHN — — CLQTZ 47 1. 000 MCRPD 100 1. 000 SARIJ 39 1. 000 TYNEP 299 1. ooo HOQOB 33 1. 000 OTTER 117 1. 000 FARBL 172 1. 000 I s o l a t e d Stream or Swamp KEOGH 45 1. 000 FARSW 75 1. 000 SPRPD 168 1. 000 SLZER 23 1. 000 DRYRN 50 1. 000 Mixed FRASR 106 0, 995 LARDC 126 0. 992 FULCR 63 1. 000 LC 40 45 1, ,000 0.000 —.,_ 0 (0) 0.000 0.000 0 (0) 0.000 0.000 0 (0) 0.Q00 0.000 0 (0) 0.000 0.000 0 (0) 0.000 0.000 0 {01 0.000 0.009 ——— 0 (2) 0.018 0.000 0 (0) 0.000 0.023 . 0 (2) 0.045 0.000 0 (0) ' 0.000 0.000 0 (01 0.000 0.000 0 (0) 0.000 0.000 0 (0) 0.000 0.000 0 (Ol- 0.000 0.000 0 COl 0.000 0.000 0 (0) 0.000 0.GQ0 0 (0) 0.000 0.000 0 (Q) 0.000 0.000 . 0 (0) 0.000 0.000 0 (0) 0.000 0.000 0.007 1 (1) 0.-014 0.135 0 (9) 0.234 0. 014 1 (1) 0.028 0. 000 0 (0) 0.000 0.000 — — — 0 (0) 0.000 0.000 0 (0) 0.000 0.000 0 (0) 0.000 0.000 0 (0) 0,000 0.000 0 (0) 0.000 0.000 0 (Q) 0,000 o.ooo 0 (Q) 0.000 0.000 — — 0 (0) ' 0.000 0.000 0 (0) 0.000 0.000 0 (0.) 0.000 o.ooo • 0 (0) 0.000 0,000 0 (0) o.ooo 0.005 1 (1) 0.010 0.008 . 0 (2) 0.0 16 0.000 ~ — 0 (0) 0.000 0.000 0 (0) 0.000 130 APPENDIX V A l l e l e frequency d i s t r i b u t i o n at the Pgi-2 l o c u s , ./Sample s i z e s are the number of f i s h (one-half the number of genes) examined. Expected numbers of heterozygotes ( i n brackets) sere c a l c u l a t e d f o r Hardy-Weinberg c o n d i t i o n s . , Population Sample S i z e A l l e l e Frequencies Heterozygotes Pgi-2*°° P g i - 2 1 * 7 Other Observed(Exp) Hetero-z y g o s i t y Marine ENGLB 30 0.983 0.017 . 1 (1) 0.033 HOBSB 37 0.986 0.014 1 (1) 0.028 WITLG 28 1.000 0.000 0 (0) 0.000 LC 19 57 0.982 0.018 2 (2) 0.035 COWIB 27 0.981 0.019 1 (D 0.037 HDEBA 30 0.950 0.050 3 (3) 0.095 SOOKP 33 0.955 0.045 . 3 (3) 0.Q86 KOKSB 68 0.919 0.081 11 (10) 0.149 SLMNC 53 0.953 0.038 0.009 3 (5) 0.090 GBAPI 41 0,915 0.073 0.012 7 (6) 0. 157 BAMFS 34 0.809 0. 191 11 (ID 0.309 SAB IE 43 0.907 0.047 0.047 8 (8) 0.173 HAINL 23 0.978 0.022 1 (1) 0.043 CONGE 44 0.909 0.091 —.- 8 (7) 0.165 SANMA 49 0.918 0.051 0.031 8 (8) 0.154 CH EH W 16 0.906 0.094 ——— 3 (3) 0. 170 Large Lake COV.IL 37 0.973 0. 014 0.014 2 (2) 0.053 SABIL 57 0.939 0.061 7 (7) 0.115 SARIN 11 0.955 0.045 , 1 (D 0.086 SPBOT 138 0.978 0.022 6 (6) 0.043 HCBTL 262 0.973 0.004 0.023 14 (14) 0.053 GBCEN 53 0.877 0. 123 11 (11) 0,216 STELL 18 0.861 0,139 5 (4) 0,239 BOBTL 21 1.000 0,000 ——— 0 (0) 0.000 Small Lake CHEML 79 0.816 0.184 —._ . 23 (24) 0.300 FOLLL 35 0. 457 0. 514 0.029 22 (18) 0.526 MAENL 75 0.773 0.227 24 (26) 0.351 LKEBB 46 0.978 0.022 — 2 (2) 0.043 HOTEL 114 0.978 0.0 22 - — 5 (5) 0.043 KLEIN 45 0.989 0.011 --- 1 (1) 0.022 PAQLK 41 1.000 0.000 0 (0) 0.000 TBOOT 50 0.990 0.010 1 (1) 0.020 GABBY 14 1.000 0.000 ., 0 (0) 0.000 BLACK 256 1.000 0.000 — 0 (0) 0.000 PATER 107 1.000 0.000 0 (0) 0.000 DEVIL 85 1. 000 0.000 0 (Q) 0.000 SOMNL 65 1 .000 0.000 — 0 (0) 0.000 HODLK 59 1.000 0.000 0 (0) 0.000 1 31 LOWBL 47 1 .000 0,000 0 (0) 0.000 MCOYL 130 0.965 0.035 9 (9) 0.068 CECIL 103 0.820 0. 180 27 (30) 0.295 MORGL 81 0.963 0.000 0.037 6 (6) 0.071 ORMND 62 1.000 0.000 0 (0) 0.000 FARWL 138 0.978 0.022 6 (6) 0.043 CEDAR 108 1.000 0.000 ——— 0 (0) 0.000 PAXTB 113 0.978 0.022 — - • 5 (5) 0.043 PAXTL 44 0.693 0.307 21 (19) 0.426 ENOSB 125 0.920 0.080 20 (18) 0. 147 ENOSL 153 0.944 0.056 — 17 (16) 0.106 GOOSE 67 0.925 0.075 10 (9) 0. 139 HECHL 37 0.986 0.014 1 in 0.028 CRANL 48 1 .000 0.000 ——— 0 (0) 0.000 Low-lying Stream or Swamp SKOOS 35 1.000 o.ooo 0 (0) 0.000 SHIPC 27 0.981 0.01 9 1 (D 0.037 LC 18 87 1.000 0.000 — 0 (0) 0.000 GIFFS 13 1.000 0.000 0 (0) 0.000 TEXAS 26 0.981 0.019 1 (1) 0.037 PAT BS 49 0.929 0.071 7 (6) 0.132 EBEYI 68 0.897 0.103 — . 14 (13) 0. 185 SKAGI 37 0.973 0.027 . 2 (2) 0.053 MOOSE 36 0.931 0.042 0.028 5 (5) 0.131 SMODL 78 1.000 0.000 0 (0) 0.000 NATHN -— — _ — . • — _ CLQTZ 47 0.702 0.277 0.021 22 (20) 0.430 MCRPD 99 0.970 0.010 0.02 0 6 (6) 0.0 59 SARIJ 39 1.000 0.000 0 (0) 0.000 TYNEP 302 0.967 0.033 20 (19) 0.075 HOQOB 33 1.000 0.000 0 (0) 0.000 OTTER 117 1 .000 0.000 0 (0) 0.000 FARBL 172 0.977 0.0 23 — — 8 (8) 0.045 I s o l a t e d S t r e a m o r Swamp KEOGH 45 1.000 0,000 — 0 (Q) 0.000 FARSW 75 1.000 0.000 0 (0) O.OOO SPRPD 168 1.000 0.000 0 (0) 0.000 SLZER 23 1.000 0.000 0 (0) 0.000 DRYRN 50 1.000 0.000 —— — 0 (0) 0.000 M i x e d FRASR 106 0.972 0. 01 9 0.009 6 (6) 0.055 LAB DC 126 0.948 0.048 0.004 11 (12) 0.099 FULCR 63 0.992 0.008 1 (D 0.016 LC 40 45 0.944 0.056 — — 5 (5) 0.106 132 APPENDIX VI A l l e l e frequency d i s t r i b u t i o n a t the Ck locus..Sample s i z e s are the number of f i s h (one-half the number of genes) examined. Expected numbers of heterozygotes ( i n brackets) were c a l c u l a t e d f o r Hardy-Weinberg c o n d i t i o n s . , P o p u l a t i o n Sample A l l e l e F r e g u e n c i e s Heterozygotes fletero-S i z e Ck»oo C k 3 5 Other Observed (Exp) z y g o s i t y Marine ENGLB 30 0.000 1.000 0 (0) 0.000 HOBS B 37 0.000 1.0 00 . 0 (0) 0.000 HITLG 33 0.015 0.985 - — 1 (1) 0.030 LC 19 57 0.000 1.000 0 (0) 0.000 COWIB 27 0.000 1.000 —^_ 0 (Q) 0.000 MDEBA — —__ . — — — " '' ' SOOKP 33 0.000 1.000 0 (Q) 0.000 KOKSB 67 0.000 1.000 , 0 (0) 0.000 SLMNC 53 0.075 0.925 6 <7> 0. 139 GBAPI 41 0.049 0.951 — 4 (4) 0.093 BAMFS 34 0.103 0.8 97 — 5 (6) 0. 185 S A l l E 44 0.080 0.920 5 (6) 0.147 HAINL 23 0. 152 0.848 7 (6) 0.258 CONGE 35 0.086 0. 9 14 — 6 (6) 0. 157 SANMA 39 0.077 0. 923 . 6 (6) 0.142 CHEHW 16 0.031 0.969 — — 1 (D 0.060 Large Lake COBIL 36 0. 000 1.000 0 (0) 0.000 SABIL 57 0.272 0.728 21 (23) 0.396 SARIN 11 0.000 1.000 0 (0) 0.0.00 SPEOT 138 0.000 0.996 0.004 1 (D 0.008 MCBTL 26 3 0.006 0. 992 0.002 4 (<•) 0.016 GBCEN 53 0.000 1.000 0 (0) 0.000 STELL 18 0. 000 1.000 0 (0) 0.000 BOBTL 21 0.000 1.000 — 0 (0) 0.000 Small Lake CHEML 83 0.000 1.000 0 (0) 0.000 FDLLL 33 0.000 1.000 — 0 (0) 0.000 HABNL 75 0.000 1.000 -•— 0 (0) 0.000 IREBE 46 0.000 1.000 —.- 0 (0) 0.000 HOTEL 114 0.018 0.982 4 (4) 0.035 KLEIN 45 0.000 1.000 0 (Q) 0.000 PAQLK 41 0.000 1.000 0 (0) 0.000 TEO UT 50 0.270 0.730 —._ 21 (20) 0.394 GABBY 14 0.000 1.000 0 (0) 0.000 BLACK 256 0.000 1.000 0 (Q) 0.000 PAT EE 107 0.000 1.000 0 (0) 0.000 DEVIL 84 0.000 1.000 , 0 (0) 0.000 SUMNL 66 0.000 1.000 —._ 0 (0) 0.000 MUDLK 59 0.000 1.000 0 (0) o.ooo 1 33 LOflRL 47 0.000 1.000 0 (0) 0.000 MCOYL 130 0. 000 1.000 0 (0) 0.000 CECIL 103 0. 000 1.000 —.._ 0 (0) o.ooo MORGL 82 0. 000 1.000 0 (Q) c.ooo ORM N D 62 0. 000 1.000 0 (0) 0.000 FAR8L 139 o.poo 1.000 — _ 0 (Q) 0.000 CEDAR 108 0.000 1.000 ——— ; 0 (0) 0.000 PAXTB 113 0.690 0.310 50 (48) 0.428 PAXTL 44 0. 148 0.852 9 (11) 0.252 ENOS B 124 0.940 0. 060 13 (14) 0.113 ENOSL 154 0.036 0.964 11 (11) 0.069 GOOSE 67 0.075 0.925 ----- 10 (9) 0. 139 HECHL 32 0. 266 0.734 13 (12) 0.390 CRANL 47 0.617 0.383 ——— 20 (22) 0.473 Low-1ying Stream or Swamp SKOOS 35 0.014 0.986 1 (1) 0.028 SHI PC 27 0.056 0.944 3 (3) 0. 106 LC 18 86 0, 663 0.337 . 38 (38) 0.447 GIFFS 13 0.077 0.923 2 (2) 0.142 TEXAS 25 0. 140 0. 860 5 (6) 0.241 PATBS 49 0.582 0.418 21 (24) 0.487 EBEYI 68 0.000 1.000 0 (0) 0.000 SKAGI 37 0.0 27 0.973 2 (2) 0.053 MOOSE 36 0.042 0.958 3 (3) 0.080 SMUDL 78 0. 538 0. 462 — 42 (39) 0.4 97 NATHN 41 0.110 0.890 9 (8) 0.196 CLQTZ 47 0.096 0.904 9 (8) 0. 174 MCBPD 99 0.066 0.934 — 13 (12) 0.123 SABIJ 35 0.386 0.614 13 (17) 0.474 TYNEP 288 0. 234 0. 766 —._ 101 (103) 0.358 HOQDB 33 0. 864 0. 136 — 9 (8) 0.235 OTTER 115 0.061 0.939 14 (13) 0.115 FARBL 172 0.003 0.997 ——— 1 (1) 0.006 I s o l a t e d Stream or Swamp KEOGH 45 0.000 1.000 • 0 (0) 0.000 FAR SB 75 0.000 1.000 - — 0 (0) 0.000 SPRPD 168 0.000 1.000 0 (Q) 0.000 SLZEB 55 0. 000 1.000 0 (Q) 0.000 DRYRN 50 0.000 1.000 — — 0 (Q) 0.000 Mixed FRA SB 106 0.009 0.991 2 (2) 0.018 LARDC 124 0.347 0.653 56 (56) 0.453 FOLCB 63 0.063 0. 937 6 (7) 0.T18 LC no 45 0.022 0.978 2 (2) 0.Q43 134 APPENDIX VII A l l e l e frequency d i s t r i b u t i o n at the Pgm l o q u s . Sample s i z e s a re the number of f i s h (one-half the number o f genes) examined. , Expected numbers of heterozygotes ( i n brackets) were c a l c u l a t e d f o r Hardy-Weinberg c o n d i t i o n s . Population Sample A l l e l e Frequencies Hetero- Hetero-S i z e Pgm l o 3Pgm 1o (>Pgffl 9 3 Pgm 9 0 Pgm 8 0 zygotes z y g o s i t y H a r i n e ENGLB 30 0.000 0. 767 0. 000 0. 200 0.033 11 (ID 0.371 HOB SB 37 0.000 0.878 0. 000 0. 108 0.014 9 (8) 0.217 HI TIG 39 0.000 0.744 0. 000 0. 244 0.013 20 (15) 0.387 IC 19 57 0.009 0. 772 0. 000 0. 219 0.000 22 (20) 0.356 COBIB 27 0.000 0.667 0. 000 0. 333 0.000 14 (12) 0.444 MDERA 22 0.023 0.750 0. 000 0. 227 0.000 9 (8) 0.385 SOOKP 32 0.0 16 0. 813 0. 000 0. 172 0.000 12 (10) 0.3 09 KOKSE 68 0.022 0.816 0. 000 0. 154 0.007 19 (21) 0.3 10 SLHNC 53 0. 198 0.575 0. ooo 0. 208 0.019 27 (31) 0.587 GRAPI 41 0.085 0.732 0. 000 0. 183 0.000 13 (17) 0.423 BAMFS 34 0.029 0. 779 0. 000 0. 176 0.000 12 (12) 0.361 SAB IE 44 0.068 0.784 0. poo 0. 136 0.011 14 (16) 0*362 HAINL 23 0.130 0.804 0. 000 0. 06 5 0.000 7 (8) 0.332 CONGE 40 0.075 0.788 0. 000 0. 125 0.000 16 (14) 0.358 SANS A 46 0.087 0.761 0. ooo 0. 152 0.000 18 (18) 0.390 CHEHW 16 o.ooo 0.719 0. 000 0. 219 0.031 , 6 (7) 0.433 Large Lake COBIL 37 o.ooo 0.689 0. 000 0. 284 0.027 13 (16) 0.444 SARIL 57 0. 158 0. 719 0. ooo 0. 123 0.000 25 (25) 0.443 SARIN 11 0.000 0.955 0. 000 0. 045 0.000 1 (1) 0.086 SPROT 138 0.0 51 0. 620 0. 000 0. 301 0.029 71 (72) 0.522 HCRTL 263 0.245 0.618 0. 023 0. 110 0.004 .125 (14 3) *0 . 545 GRCEN 51 0.373 0. 529 0. 010 0. 078 0.010 26 (29) 0.575 STELL 18 0.028 0.694 0. 000 0. 278 0.000 7 (8) 0.440 EOBTL 21 0.000 0.976 0. ooo 0. 024 0.000 1 CD 0.047 Small Lake CHEML 83 0.831 0.000 0. 000 0. 169 0.0 00 26 (23) 0.281 FOLLL 33 0. 121 0. 394 0. 000 0. 485 0.000 20 (20) 0.595 MARNL 75 0.833 0.020 0. 000 0. 147 0.000 25 (21) 0.284 LKESR 46 0.087 0.793 0. 000 0. 120 0.000 15 (16) 0.349 HOTEL 114 0.395 0.048 0. 000 0. 557 0.000 59 (61) 0.531 KLEIN 45 0.300 0.489 0. ooo 0. 133 0.078 32 (29) 0.647 PAQLK 41 0.000 0.622 0. 000 0. 378 0.000 17 (19) 0.4,70 TROUT 50 0.000 0.630 0. 000 0. 370 o.ooo 25 (23) 0.466 GARBY 14 0.464 0.321 0. 000 0. 214 0.000 12 (9) 0.636 BLACK 255 0. 143 0. 288 0. 535 0. 033 0.000 139 (155) 0.609 PATER 107 0.397 0.383 0. 220 0. 000 0.0 00 72 (69) 0.647 DEVIL 85 0.00 0 1.000 0. 000 0. 000 0.000 0 (0) 0.000 SUMNL 66 0.455 0.288 0. 053 0. 205 0.000 41 (44) 0.665 MUDLK 57 0.272 0.72 8 0. 000 0. 000 0.000 23 (23) 0.396 135 LOB EL 47 0.000 0.986 MCOYL 130 0.000 0.946 CECIL 103 0.000 0. 704 HOEGL 80 0.22 5 0.775 OBMND 62 0.000 0.855 FARWL 138 0.094 0.254 CEDAE 108 0. 148 0.593 PAXTB 93 0.935 0.065 PAXTL 44 0.239 0.716 ENOS B 62 0. 40 3 0. 573 ENOSL 154 0.010 0.558 GOOSE 65 0.346 0.000 HECHL 37 0.068 0. 378 CBANL 47 0.000 -0.628 Low-lying Stream or Swamp SKOOS 34 0.000 0.897 SHI PC 27 0.000 0.796 LC 18 87 0.552 0. 448 GIFFS — — _ TEXAS 25 0. 180 0.720 PATBS 49 0.418 0.214 EBEYI 68 0,074 0.618 SKAGI 37 0. 216 0. 581 MOOSE 36 0.125 0.639 SMUDL 78 0.6 54 0. 346 NATHN 41 0.378 0.427 CLQTZ 47 0.149 0- 734 MCBPD 100 0, 260 0.605 SABIJ 35 0. 143 0.800 TYNEP 323 0.054 0.553 HOQBB 33 0.000 0.833 OTTEB 117 0. 150 0.812 FABBL 172 0.0 58 0, 230 I s o l a t e d S t r e a to or Swamp KEOGH 45 0.433 0. 322 FABSH 71 o.poo 0.767 SPBPD 168 0.003 0.988 SLZER 61 0.000 0.295 DBYBN 50 0.0 00 1.000 Mixed FE A SB 105 0. 143 0.714 IABDC 112 0.362 0.554 FOLCB 63 0. 0 16 0. 516 LC 40 42 0.071 0.702 0.000 0. 032 0.000 3 (3) 0.0 27 0.000 0. 050 0.000 14 (13) 0. 103 0.000 0. 117 0. 180 46 (47) 0.458 0.000 0. 000 0.000 30 (28) 0.349 0. 145 0. 000 0.000 16 (15) 0.248 0.525 0. 127 0.000 83 (88)**0.635 0. 106 0. 153 0.0 00 67 (64) 0.592 0.000 0. 000 0.000 12 (11) 0.122 0.000 0. 045 0.000 17 (19) 0.428 0.000 0. 024 0.000 30 (32) 0.509 0.000 0. 432 0.000 77 (77) 0.502 0.000 0. 654 0.000 25 (29) 0.453 0. 000 0. 486 0.068 22 (23) 0,612 0.000 0. 181 0.000 31 (25) 0.536 0.000 0. 10 3 0.000 5 (6) 0.185 0.000 0.185 0.019 7 (9) 0.332 0.000 o.ooo 0.000 40 (43) 0.495 0. 000 0. 100 0. 0 00 10 (11) 0.439 0.000 0.367 0.000 31 (31) 0.645 0.000 0.30 1 0.000 33 (35) 0.522 0.000 0. 176 0.027 20 (22) 0.584 0,000 0.222 0.000 22 (19) 0.527 0.000 o.ooo O.OOO 30 (35) 0.453 0.024 0. 171 0.000 27 (26) 0.645 0.000 0.117 0.000 20 (20) 0. 425 0. 025 0. 110 0.000 58 (55) 0.554 0.000 0.057 0.000 9 (12) 0.336 0.000 0.393 0-000 166(173) 0.537 0,000 0,167 0.000 9 (9) 0,278 0.000 0.038 0.000 36 (37) 0.317 0. 581 0. 131 0.000 97(101) 0.589 0.000 0.244 o.ooo 27 (29) 0.649 0. 227 0.007 0.000 27 (26) 0.360 0.000 0.009 0.000 4 •- (».)• 0.0 24 0.000 0. 697 0.000 26 (26) 0.427 0,000 O.POO 0.0 00 0 (P) : 0.000 0,010 0. 133 0. 000 44 (47) 0.452 0.000 0.071 0.009 59 (62) 0.557 o. poo 0.46 8 0.000 28 (32) 0.514 0.000 0.226 0.000 17 (19) 0.451 * P < 0.05 ** P < 0.01 1 36 APPENDIX VIII Expected mean squares and components of varia n c e f o r nested a n a l y s e s of v a r i a n c e performed on transformed gene f r e q u e n c i e s of Bear and Somass r i v e r system p o p u l a t i o n s . T o t a l s and percentages c a l c u l a t e d f o r components of variance do not i n c l u d e negative v a l u e s . Source of V a r i a b i l i t y Expected Hean Square Systems S 2 ( e r r ) + 1.742S 2(loc) • 6. 543S 2 (hab) .* 14. 733S 2 (sys) H a b i t a t s S 2 (err) • 1.727S 2(loc) + 4. 181S 2 (hab) L o c a t i o n s S 2 ( e r r ) + 1.783S 2(loc) E r r o r S 2 (err) Components of Variance Mdh-1 Mdh-3 Pgi-2 Pgm* oo Pg m9 3 s 2 (err) .002 .003 ,002 .007 .00 3 (8$) (2%) (20%) (6%) (3%) s 2 (loc) .0 10 .104 .006 . 104 .070 (40%) (53%) (60%) (84%) (67%) s 2 (hab) .013 .022 .002 -.006 -.001 {52%) (11%) (20%) s 2 (sys) -. 006 .067 -.002 .013 .032 (34%) (10%) (30%) T o t a l .025 .196 .010 . 124 . 105 

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