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Isolating mechanisms and speciation in Gasterosteus aculeatus L. Hagen, Don Warren 1966

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ISOLATING MECHANISMS AND SPECIATION IN GASTEROSTEUS ACULEATUS L . by DON WARREN HAGEN B . A . , U n i v e r s i t y o f Texas , 1957 M „ A 0 / U n i v e r s i t y o f Texas , 1963 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department o f Z o o l o g y We a c c e p t t h i s t h e s i s as c o n f o r m i n g to the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA May, 1966 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x -t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s m a y b e g r a n t e d b y t h e H e a d o f my D e p a r t m e n t o r b y h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n -c i a l g a i n s h a l l n o t b e a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, C a n a d a D a t e The University of B r i t i s h Columbia FACULTY OF GRADUATE STUDIES PROGRAMME OF THE FLNAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY of DON WARREN HAGEN B.A., University of Texas, 1957 M.A., University of Texas, 1963 TUESDAY, JULY 5, 1966, AT 3:30 P.M. IN ROOM 3332, BIOLOGICAL SCIENCES BUILDING COMMITTEE IN CHARGE Chairman: D..H. Copp J. F. Bendell, Do H. Chitty W. S. Hoar G. C. Hughes C. C. Lindsey J. D. McPhail External Examiner: P. M. Sheppard The University, Liverpool England Supervisor: C. C. Lindsey ISOLATING MECHANISMS AND SPECIATION IN GASTEROSTEUS ACULEATUS L. ABSTRACT Intensive f i e l d observations were conducted on a small B r i t i s h Columbia coastal stream for two years? supplemented by laboratory rearing and experiment. Stress was placed upon possible i s o l a t i n g mechanismss. as outlined by Mayr, that might serve to keep the marine stickleback (trachurus) and the freshwater form ( l e i u r u s ) d i s t i n c t . Lelurus permanently occupies the upper reaches of the stream; trachurus i s anadromous, but. enters the lower reaches to breed i n "pure" fresh-water. Between the breeding ground of the two, where some coexistence occurs, hybridization between leiurus and trachurus i s extensive, but it: i s r e s t r i c t e d to very narrow zones both i n L i t t l e Campbell River near Vancouver and i n certain Vancouver Island streams. Morphological analysis provides firm circumstantial evidence that hybrids are p l e n t i f u l and that back-crossing occurs. Hybridization i s confirmed by rearing experiments i n the laboratory with crosses i n a l l com-binations. Most backcrossing i s to the freshwater race and this i s considered i n d e t a i l . Behavioural experiments demonstrated the absence of mate preference; random mating occurs with respect to ethological i s o l a t i o n and hybrids perform courtship and parental care a c t i v i t i e s normally. Rearing hybrids uniformly and i n a l l conbinations demonstrated the absence of genetic incompatibility, and vigorous offspring were produced. F e r t i l e hybrids are abundant i n the wild, but the sexes are seasonally isolated from one another. Behavoiral and genetic blocks to hybridization are absent, and thus there i s no means to prevent hybridiza-tion where leiurus and trachurus come together. But evidence.from the f i e l d and from preference tests demon-strated that ecological i s o l a t i n g mechanisms are very powerful barriers to hybridization. This i s enforced to some extent: by p a r t i a l seasonal i s o l a t i o n , but early spawning migrants make a major contr .bution to hybridiza-tion i n the L i t t l e Campbell River. Several factors probably operate to cause very narrow hybrid zones in. stickle-backs, including, a sedentary habit, habitat pre-ference, and hybrid i n f e r i o r i t y outside the hybrid zone. The l a t t e r could not be detected inside the hybrid zone. The complex of Isolating mechanisms found may well, pro-duce stable hybrid zones over long periods of time, and i s discussed o Reinforcement of ecological. 1 sol at ing mechanisms probably occurs, and Moore's c r i t i c i s m of the spread of reinforced genotypes would not: apply i n this instance. Adaptation and consequent, ecological i s o l a t i n g mechanisms are the cause of hybrid i n f e r i o r i t y . In this instance pre and postmating mechanisms cannot: be'separa-ted, for they go hand i n hand. 1 Geographic speci.atlon. i s possible but problematical, and sympatric speciation cannot be excluded as a plausible explanation of divergence between sticklebacks as a result of disruptive selection. D i s t r i b u t i o n a l patterns of leiurus can best be explained by repeated and indepen-dent origins from trachurus. The two forms f u l f i l l the species d e f i n i t i o n by Mayr and remain reproductively isolated, have well de-veloped i s o l a t i n g mechanisms, and show considerable gene-t i c divergence, much of i t polygenic. There i s no evi-dence that, introgression occurs, but a reverse c l i n e i n leiurus as well as very narrow hybrid zones demonstrates that selection severely r e s t r i c t s gene flow. Reproduc-ti v e i s o l a t i o n between marine and freshwater forms seems to be widespread around the hemisphere. GRADUATE STUDIES Fie l d of Study: Zoology Advanced Ichthyology C. C. Lindsey N. J. Wilimovsky Co V. Finnegan P. Ford D, Suzuki D. Chitty C e l l Biology Seminar Advanced Ecology PUBLICATIONS Hagen, D.W. Evidence of adaptation to environmental temperatures in three species of Gambusia. S. W. Nat. 9(1):6 Reproductive behavior of Gambusia heterochi Copeia, 1957:289 i C h a i r m a n : D r , C . C . L i n d s e y ABSTRACT I n t e n s i v e f i e l d o b s e r v a t i o n s were c o n d u c t e d on a s m a l l B r i t i s h Columbia c o a s t a l s t ream f o r two y e a r s , supplemented by l a b o r a t o r y r e a r i n g and e x p e r i m e n t . S t r e s s was p l a c e d upon p o s s i b l e i s o l a t i n g mechanisms, as o u t l i n e d by Mayr, t h a t might s e r v e to keep the marine s t i c k l e b a c k ( t r a c h u r u s ) and the f r e s h w a t e r form ( l e i u r u s ) d i s t i n c t . L e i u r u s p e r m a n e n t l y o c c u p i e s the upper reaches o f the stream? t r a c h u r u s i s anadromous, b u t e n t e r s the lower reaches to b r e e d i n " p u r e " f r e s h w a t e r . Between the b r e e d i n g ground o f the two, where some c o e x i s t e n c e o c c u r s , h y b r i d i z a t i o n between l e i u r u s and t r a c h u r u s i s e x t e n s i v e , b u t i t i s r e s t r i c t e d to v e r y narrow zones b o t h i n L i t t l e Campbel l R i v e r near Vancouver and i n c e r t a i n Vancouver I s l a n d s t r e a m s . M o r p h o l o g i c a l a n a l y s i s p r o v i d e s f i r m c i r c u m -s t a n t i a l e v i d e n c e t h a t h y b r i d s are p l e n t i f u l and t h a t b a c k -c r o s s i n g o c c u r s . H y b r i d i z a t i o n i s c o n f i r m e d by r e a r i n g exper iments i n the l a b o r a t o r y w i t h c r o s s e s i n a l l c o m b i n a t i o n s . Most b a c k c r o s s i n g i s to the f r e s h w a t e r race and t h i s i s c o n s i d e r e d i n d e t a i l . B e h a v i o u r a l exper iments demonstra ted the absence o f mate p r e f e r e n c e ; random m a t i n g o c c u r s w i t h r e s p e c t to e t h o l o g i c a l i s o l a t i o n and h y b r i d s p e r f o r m c o u r t s h i p and p a r e n t a l c a r e a c t i v i t i e s n o r m a l l y . R e a r i n g h y b r i d s u n i f o r m l y and i n a l l c o m b i n a t i o n s demonstrates the absence of g e n e t i c i n c o m p a t i b i l i t y , and v i g o r o u s o f f s p r i n g were produced, , F e r t i l e h y b r i d s are abundant i n the w i l d , b u t the sexes are s e a s o n a l l y i s o l a t e d from one a n o t h e r . B e h a v i o u r a l and g e n e t i c b l o c k s to h y b r i d i z a t i o n are a b s e n t , and thus t h e r e i s no means to p r e v e n t h y b r i d i z a t i o n where l e i u r u s and t r a c h u r u s come t o g e t h e r . But e v i d e n c e from the f i e l d and from p r e f e r e n c e t e s t s demonstrates t h a t e c o l o g i c a l i s o l a t i n g mechanisms are v e r y p o w e r f u l b a r r i e r s to h y b r i d i z a t i o n . T h i s i s e n f o r c e d to some e x t e n t by p a r t i a l s e a s o n a l i s o l a t i o n , but. e a r l y spawning m i g r a n t s make a major c o n t r i b u t i o n to h y b r i d i z a t i o n i n the L i t t l e Campbel l R i v e r . S e v e r a l f a c t o r s p r o b a b l y o p e r a t e to cause v e r y narrow h y b r i d zones i n s t i c k l e -b a c k s , i n c l u d i n g a s e d e n t a r y h a b i t , h a b i t a t p r e f e r e n c e , and h y b r i d i n f e r i o r i t y o u t s i d e the h y b r i d z o n e . The l a t t e r c o u l d not be d e t e c t e d i n s i d e the h y b r i d z o n e . The' complex o f i s o l a t i n g mechanisms f o u n d may w e l l produce s t a b l e h y b r i d zones o v e r l o n g p e r i o d s o f t i m e , and i s d i s c u s s e d . R e i n f o r c e m e n t o f e c o l o g i c a l i s o l a t i n g mechanisms p r o b a b l y o c c u r s , and M o o r e ' s c r i t i c i s m o f the s p r e a d o f r e i n f o r c e d genotypes would not a p p l y i n t h i s i n s t a n c e . A d a p t a t i o n and consequent e c o l o g i c a l i s o l a t i n g mechanisms are the cause o f h y b r i d i n f e r i o r i t y . In t h i s i n s t a n c e pre and p o s t m a t i n g mechanisms cannot be s e p a r a t e d , f o r they go hand i n h a n d . G e o g r a p h i c s p e c i a t i o n i s p o s s i b l e b u t p r o b l e m a t i c a l , and s y m p a t r i c s p e c i a t i o n cannot be e x c l u d e d as a p l a u s i b l e e x p l a n a t i o n of d i v e r g e n c e between s t i c k l e b a c k s as a r e s u l t o f d i s r u p t i v e s e l e c t i o n . D i s t r i b u t i o n a l p a t t e r n s o f l e i u r u s c a n best be ex p l a i n e d by repeated and independent o r i g i n s from trachurus. The two forms f u l f i l l the species d e f i n i t i o n by Mayr and remain r e p r o d u c t i v e l y i s o l a t e d , have w e l l developed i s o l a t i n g mechanisms, and show considerable genetic divergence, much of i t p o l y g e n i c . There i s no evidence that i n t r o g r e s s i o n occurs, but a reverse c l i n e i n l e i u r u s as w e l l as very narrow h y b r i d zones-demonstrates that s e l e c t i o n s e v e r e l y r e s t r i c t s gene flow. Reproductive i s o l a t i o n between marine and freshwater forms seems to be widespread around the hemisphere. i v TABLE OF CONTENTS PAGE INTRODUCTION 1 THE STUDY AREA 5 MATERIALS AND METHODS 5 The H a b i t a t 6 The H y b r i d Zone 13 MORPHOLOGICAL ANALYSIS 15 MATERIALS AND METHODS 15 A l l o p a t r i c P o p u l a t i o n s 18 The H y b r i d Zone 20 The H y b r i d Index 24 S c o r i n g and W e i g h t i n g 27 L a b o r a t o r y Reared F i s h 29 V a r i a t i o n W i t h i n L e i u r u s 36 SEASONAL ISOLATION 38 MATERIALS AND METHODS 38 RESULTS 39 H y b r i d s 41 E a r l y Spawning T r a c h u r u s 43 Vancouver I s l a n d P o p u l a t i o n s 45 ETHOLOGICAL ISOLATION 48 MATERIALS AND METHODS 48 RESULTS 51 Mate P r e f e r e n c e T e s t s 51 Female C h o i c e T e s t s - 53 H y b r i d R e p r o d u c t i v e A c t i v i t i e s 54 ECOLOGICAL ISOLATION 5 6 DISPERSAL AND TRANSFER EXPERIMENTS 5 6 M a t e r i a l s and Methods 56 R e s u l t s 5 7 D i s p e r s a l Exper iments 5 7 T r a n s f e r Exper iments 58 PREFERENCE TESTS 66 M a t e r i a l s and Methods 67 S u b s t r a t e P r e f e r e n c e T e s t s 67 V e g e t a t i o n P r e f e r e n c e T e s t s 68 C u r r e n t P r e f e r e n c e T e s t s 68 Water Q u a l i t y P r e f e r e n c e T e s t s 69 R e s u l t s 70 S u b s t r a t e P r e f e r e n c e T e s t s 70 V e g e t a t i o n P r e f e r e n c e T e s t s 71 C u r r e n t P r e f e r e n c e T e s t s 72 Water Q u a l i t y P r e f e r e n c e T e s t s 72 ECOLOGICAL OBSERVATIONS ON VANCOUVER ISLAND 74 NESTING SITES AND MATERIALS 76 TROPHIC ADAPTATIONS 78 V PAGE POSTMATING ISOLATING MECHANISMS 81 MATERIALS AND METHODS 81 RESULTS 83 S u r v i v a l to H a t c h i n g 83 S u r v i v a l F r y 85 H y b r i d F e c u n d i t y 88 H y b r i d I n f e r i o r i t y 90 DISCUSSION 91 ISOLATING MECHANISMS AND HYBRIDIZATION 91 I s o l a t i n g Mechanisms 91 The H y b r i d Zone 97 SPECIATION 105 TAXONOMIC STATUS OF LEIURUS AND TRACHURUS 114 CONCLUSIONS 117 LITERATURE CITED 119 v i L I S T OF TABLES TABLE PAGE I A s y n o p s i s o f h a b i t a t , c h a r a c t e r i s t i c s i n the L i t t l e Campbel l R i v e r . P h y s i c a l and c h e m i c a l d a t a r e p r e s e n t averages f rom May t h r o u g h A u g u s t , 9 I I D e n s i t y e s t i m a t e s f o r l e i u r u s and t r a c h u r u s from s e v e r a l h a b i t a t s i n the L i t t l e Campbel l R i v e r , Each f i g u r e r e p r e s e n t s the average c a t c h from t h r e e a d j a c e n t 15 f o o t s e i n e h a u l s . C o n s e c u t i v e f i g u r e s f o r a g i v e n date are s i m i l a r e s t i m a t e s made a t 200 f o o t i n t e r v a l s o f s t r e a m . D e n s i t i e s o f l e i u r u s i n 1965, not g i v e n , were l i k e those f o r 1964, 11 I I I Numbers o f b r e e d i n g h y b r i d s t a k e n on dates i n d i c a t e d , 42 IV P r o p o r t i o n o f each r a c e b r e e d i n g i n Vancouver I s l a n d s t reams , 1965, 46 V R e s u l t s o f mate p r e f e r e n c e t e s t s (above) and female p r e f e r e n c e t e s t s (below) t a k e n from C o l e ' s c l o s e d s e q u e n t i a l t e s t d e s i g n , 52 VI P r e f e r e n c e t e s t s w i t h s t ream w a t e r . Numbers o f f i s h i n each compartment ( tea c o l o u r e d , c l e a r ) a t i n d i c a t e d t imes f o l l o w i n g i n t r o d u c t i o n to a p p a r a t u s . 73 V I I H a t c h i n g s u c c e s s o f h y b r i d c r o s s e s . Numbers i n p a r e n t h e s i s r e p r e s e n t p l a t e c o u n t s o f p a r e n t s . C o n t r o l s p r e s e n t e d i n l e f t hand columns and c o r r e s p o n d i n g h y b r i d c r o s s e s i n r i g h t hand 84 c o l u m n , a - g V I I I S u r v i v a l o f l a b o r a t o r y r e a r e d h y b r i d s w i t h 86 c o n t r o l s p r e s e n t e d i n l e f t hand c o l u m n s . a - b IX Numbers o f mature eggs f o r h y b r i d s and f o r each r a c e . Sample s i z e o f 40 f o r each r a c e and f o r h y b r i d s . 89 V l l L I S T OF FIGURES FIGURE , PAGE 1 . L o c a t i o n o f c o l l e c t i n g S t a t i o n s on the L i t t l e Campbel l R i v e r . 7 2. D i s t r i b u t i o n o f r a c e s and h y b r i d s o f s t i c k l e -back a t S t a t i o n s on the L i t t l e Campbel l R i v e r . Data b a s e d on more than 6000 f i s h . M i n i m a l sample s i z e f o r S t a t i o n was 200. 10 3 . L a t e r a l p l a t e counts f o r a l l o p a t r i c p o p u l a t i o n s o f l e i u r u s and t r a c h u r u s and o f h y b r i d s , 19 4 . G i l l r a k e r c o u n t s f o r a l l o p a t r i c p o p u l a t i o n s o f l e i u r u s and t r a c h u r u s and o f h y b r i d s „ 21 5 . E l e c t r o p h o r e t i c b a n d i n g p a t t e r n s f o r a l l o p a t r i c p o p u l a t i o n s o f l e i u r u s and t r a c h u r u s and o f h y b r i d s . D i a g n o s t i c bands of each r a c e are i n d i c a t e d b y a r r o w s . Sample s i z e s are 60 l e i u r u s ; 60 t r a c h u r u s ? 120 h y b r i d s . W i t h i n the h y b r i d c l a s s i n d i v i d u a l s h a d e i t h e r b o t h bands d i a g n o s t i c f o r each r a c e p r e s e n t o r one band d i a g n o s t i c f o r one o r the o t h e r p r e s e n t . These may be i n t e r p r e t e d as e i t h e r Fi h y b r i d s o r e i t h e r b a c k c r o s s c l a s s . Percentages f o r the t h r e e c l a s s e s were t 11.8% F ^ ; 80.2% b a c k c r o s s e s to l e i u r u s ? 8% b a c k c r o s s e s to t r a c h u r u s . 22 6. M o r p h o l o g i c a l c h a r a c t e r i s t i c s f o r a l l o p a t r i c p o p u l a t i o n s o f l e i u r u s and t r a c h u r u s and o f h y b r i d s . 23 7„ F r e q u e n c y d i s t r i b u t i o n o f the h y b r i d i n d e x . I n v e r t e d h i s t o g r a m s r e p r e s e n t a l l o p a t r i c p o p u l a t i o n s ; u p r i g h t h i s t o g r a m s r e p r e s e n t samples from the h y b r i d z o n e . 30 8. L a t e r a l p l a t e counts f o r l a b o r a t o r y r e a r e d ( b l a c k h i s t o g r a m s ) and o f w i l d caught f i s h (hatched h i s t o g r a m s ) . L e i u r u s u s e d as p a r e n t s to o b t a i n o f f s p r i n g h a d p l a t e counts from 3-5 ; t r a c h u r u s counts 3 2 - 3 4 „ Data b a s e d on 6 i n t r a r a c i a l c r o s s e s w i t h l e i u r u s p a r e n t s , 6 i n t r a r a c i a l c r o s s e s w i t h t r a c h u r u s p a r e n t s , 7 i n t e r r a c i a l c r o s s e s f o r F \ h y b r i d s , and 7 b a c k c r o s s e s . A p p r o x i m a t e l y e q u a l numbers o f r e c i p r o c a l c r o s s e s made. 31 VXX1 FIGURE PAGE 9. G i l l r a k e r counts f o r l a b o r a t o r y r e a r e d ( b l a c k h i s t o g r a m s ) and o f w i l d caught (hatched h i s t o g r a m s ) f i s h p r e s e n t e d f o r c o m p a r i s o n s . See c a p t i o n , F i g u r e 8, f o r o t h e r d e t a i l s . 33 10. Body depth i n t o s t a n d a r d l e n g t h o f l a b o r a t o r y r e a r e d f i s h . See c a p t i o n , F i g u r e 8, f o r o t h e r d e t a i l s . , 35 11. C l i n a l v a r i a t i o n i n numbers o f p l a t e s o f l e i u r u s . Data are b a s e d on counts o f more than 2000 f i s h . Samples of a t l e a s t 200, and u s u a l l y 400-500 f i s h , were made a t each S t a t i o n . 37 12. B r e e d i n g seasons f o r the r a c e s o f s t i c k l e b a c k i n the L i t t l e Campbel l R i v e r , 1964, Data f o r 1965; f i r s t , b r e e d i n g l e i u r u s , March 2 and f o r t r a c h u r u s May 12; f i r s t f r y o f l e i u r u s , A p r i l 28 and f o r t r a c h u r u s J u l y 7; peak o f b r e e d i n g f o r l e i u r u s , A p r i l 12-26 and f o r t r a c h u r u s June 6 - A u g u s t 2; end o f season f o r l e i u r u s , J u l y 10 and f o r t r a c h u r u s , September 18. 40 13. Recapture d a t a f o r l e i u r u s t r a n s f e r r e d from Pond D to S t a t i o n D . 59 14. Recapture d a t a f o r the t r a n s f e r o f l e i u r u s from S t a t i o n D to S t a t i o n N . Zero r e p r e s e n t s p o i n t o f r e l e a s e . See t e x t f o r f u r t h e r d e t a i l s . 61 15. Recapture d a t a f o r l e i u r u s t r a n s p l a n t e d from S t a t i o n D to S t a t i o n N . Zero r e p r e s e n t s p o i n t o f r e l e a s e , 63 16. Recapture d a t a f o r t r a c h u r u s t r a n s p l a n t e d from S t a t i o n N to S t a t i o n D . Zero r e p r e s e n t s p o i n t o f r e l e a s e , 65 17. Food i tems t a k e n from stomachs o f l e i u r u s and t r a c h u r u s , See t e x t f o r e x p l a n a t i o n . 80 i x ACKNOWLEDGEMENTS I am g r a t e f u l to D r . C . C . L i n d s e y i n more ways than I can t e l l . Many o f the t e c h n i q u e s and i d e a s u s e d i n t h i s r e s e a r c h d e v e l o p e d through d i s c u s s i o n s w i t h h i m , and I would e s p e c i a l l y thank him f o r a l l o w i n g me to f r e e l y choose my r e s e a r c h and f o r h i s i n s p i r a t i o n . To the Vancouver P u b l i c A q u a r i u m I s h o u l d l i k e to e x p r e s s my thanks f o r the use o f l a b o r a t o r y f a c i l i t i e s . I a l s o e x t e n d my s i n c e r e a p p r e c i a t i o n to a l l those who h e l p e d me p u l l a s e i n e , and e s p e c i a l l y M r . P e t e r Johansen, M r . A l e x Peden and M r . Ken P e t r i e . I am g r a t e f u l to M r . P e t e r E l l i c k s o n f o r h i s a s s i s t a n c e i n and around the museum and f o r i m p r o v i n g my mood. D r . D0 E . M c A l l i s t e r , C u r a t o r of F i s h e s , N a t i o n a l Museum o f Canada, v e r y k i n d l y s u p p l i e d me w i t h m u c h . d a t a c o n c e r n i n g the d i s t r i b u t i o n o f s t i c k l e b a c k s . I thank D r s : N . R. L i l e y , D„ S u z u k i , and J . T . McFadden f o r the p r o f i t a b l e d i s c u s s i o n s I have had w i t h them. F o r t h e i r d i s c u s s i o n s , s u g g e s t i o n s , and e n t h u s i a s m , I must e s p e c i a l l y thank D r s : Dennis C h i t t y and J . D„ M c P h a i l . Most o f a l l I am g r a t e f u l to my p a r e n t s f o r t h e i r encouragement . INTRODUCTION The foremost o b j e c t i v e o f t h i s r e s e a r c h was to determine which i s o l a t i n g mechanisms, i f any, s e p a r a t e the marine and f r e s h w a t e r forms o f the t h r e e - s p i n e d s t i c k l e b a c k , G a s t e r o s t e u s a c u l e a t u s L . A second purpose was to i n t e r p r e t the p r o c e s s o f s p e c i a t i o n t h a t gave r i s e to the two f o r m s . F i n a l l y , an at tempt was made to r e s o l v e t h e i r taxonomic r e l a t i o n s h i p s . I s o l a t i n g mechanisms have been a c t i v e l y i n v e s t i g a t e d e v e r s i n c e Dobzhansky (1937), Mayr (1942), and H u x l e y (1942) emphasized t h e i r i m p o r t a n c e . Today i t i s w e l l e s t a b l i s h e d t h a t i s o l a t i n g mechanisms are among the most i m p o r t a n t a t t r i b u t e s o f s p e c i e s s i n c e these p r o p e r t i e s have p e r m i t t e d the vast^ number o f s p e c i e s t h a t now e x i s t . E s p e c i a l l y i m p o r t a n t are s t u d i e s o f i s o l a t i n g mechanisms between c l o s e l y r e l a t e d s y m p a t r i c s p e c i e s . Indeed, a knowledge o f these mechanisms i s e s s e n t i a l to u n d e r s t a n d i n g the o r i g i n o f s p e c i e s The t h r e e - s p i n e d s t i c k l e b a c k o f f e r s e x c e p t i o n a l o p p o r t u n i t y f o r such i n v e s t i g a t i o n s . Both the marine and f r e s h w a t e r form of t h i s f i s h i s w i d e s p r e a d i n b o r e a l r e g i o n s . I t i s e a s y to m a i n t a i n i n the l a b o r a t o r y and t h i s t o g e t h e r w i t h i t s abundance makes i t an i d e a l a n i m a l f o r f i e l d and l a b o r a t o r y r e s e a r c h . F u r t h e r m o r e , t h e r e i s a l r e a d y c o n s i d e r -a b l e i n f o r m a t i o n a v a i l a b l e on the s t i c k l e b a c k . The p e r t i n e n t l i f e h i s t o r y , which p r o v i d e s the f o u n d a t i o n and o r i e n t a t i o n o f t h i s r e s e a r c h , i s r e v i e w e d below 2 There are two d i s t i n c t forms o f s t i c k l e b a c k . One e x i s t s i n the sea f o r most o f i t s l i f e ; the o t h e r remains i n f r e s h w a t e r . F o l l o w i n g the t e r m i n o l o g y o f Munzing (1963) and o t h e r s the marine form i s r e f e r r e d to as t r a c h u r u s and the f r e s h w a t e r form as l e i u r u s , and f o r c o n v e n i e n c e they are c a l l e d r a c e s . T r a c h u r u s l i v e s i n the sea f o r most o f the y e a r , and i n the s p r i n g i t m i g r a t e s i n t o f r e s h w a t e r streams or r i v e r s to b r e e d . In e a r l y autumn o f f s p r i n g and a d u l t s r e t u r n to the s e a . The l i f e span i s p r o b a b l y one year (van Mullem and v a n der V l u g t , 1964) . T r a c h u r u s i s e a s i l y i d e n t i f i e d by a complete s e r i e s o f bony p l a t e s a l o n g i t s s i d e s , l a r g e s i z e , a t e r e t e body shape, l o n g d o r s a l and p e l v i c s p i n e s , few v e r t e b r a e , and a b r i g h t s i l v e r c o l o u r . The f r e s h w a t e r form, l e i u r u s , l i v e s i n f r e s h w a t e r streams and ponds throughout i t s l i f e . I t i s d i s t i n g u i s h e d from t r a c h u r u s by i t s v e r y few l a t e r a l bony p l a t e s , s m a l l s i z e , chunky body shape, s h o r t d o r s a l and p e l v i c s p i n e s , more v e r t e b r a e , and an o l i v e c o l o u r , m o t t l e d w i t h i n d i s t i n c t b a r s on the b a c k . The males o f b o t h forms assume b r i l l i a n t n u p t i a l c o l o u r s w i t h r e d t h r o a t s and b l u e i r i s e s . D u r i n g the b r e e d i n g season they are v e r y a g g r e s s i v e and e s t a b l i s h t e r r i t o r i e s , b u i l d n e s t s i n the s u b s t r a t e , g u a r d and f a n t h e i r eggs, and e x h i b i t p a r e n t a l c a r e o f f r y . L e i u r u s and t r a c h u r u s are t y p i c a l l y s y m p a t r i c d u r i n g the b r e e d i n g season and presumably h y b r i d i z e (Munzing, 1963) so t h a t one i s l e d to ask how the two m a i n t a i n t h e i r i d e n t i t y . There are a h o s t o f mechanisms t h a t may i s o l a t e 3 c l o s e l y r e l a t e d s p e c i e s f rom one a n o t h e r , b u t d e s p i t e t h e i r importance v e r y few at tempts have been made to s y s t e m a t i c a l l y determine w h i c h o f the p o t e n t i a l i s o l a t i n g mechanisms f u n c t i o n f o r a g i v e n p a i r o f s p e c i e s . Such s t u d i e s are n e c e s s a r y , however, f o r they demonstrate the r e l a t i v e importance o f the mechanisms and the i n t e r p l a y among them. The f o l l o w i n g c l a s s i f i c a t i o n o f i s o l a t i n g mechanisms d e v e l o p e d by Mayr (1963) i s u s e d i n t h i s i n v e s t i g a t i o n : 1, Mechanisms t h a t p r e v e n t i n t e r s p e c i f i c c r o s s e s (premating mechanisms) , (a) P o t e n t i a l mates do not meet ( s e a s o n a l and h a b i t a t i s o l a t i o n ) , (b) P o t e n t i a l mates meet b u t do not mate ( e t h o l o g i c a l i s o l a t i o n ) . (c) C o p u l a t i o n a t t e m p t e d b u t no t r a n s f e r o f sperm takes p l a c e (mechanica l i s o l a t i o n ) , 2, Mechanisms t h a t reduce f u l l s u c c e s s o f i n t e r s p e c i f i c c r o s s e s (postmat ing mechanisms) , (a) Sperm t r a n s f e r takes p l a c e b u t egg i s not f e r t i l i z e d (gametic m o r t a l i t y ) , (b) Egg i s f e r t i l i z e d b u t zygote d i e s (zygote m o r t a l i t y ) , (c) Zygote produces an F-^  h y b r i d o f r e d u c e d v i a b i l i t y ( h y b r i d i n v i a b i l i t y ) „ (d) F-j_ h y b r i d zygote i s f u l l y v i a b l e b u t p a r t i a l l y o r c o m p l e t e l y s t e r i l e , o r produces d e f i c i e n t F^ ( h y b r i d s t e r i l i t y ) , 4 S t i c k l e b a c k s are e x t e r n a l f e r t i l i z e r s so that mechanical i s o l a t i o n can be ommitted from f u r t h e r c o n s i d e r a t i o n . The i n t e n t throughout the research has been to examine the r e l a t i v e importance of the p o s s i b l e i s o l a t i n g mechanisms l i s t e d above combining i n t e n s i v e f i e l d and l a b o r a t o r y i n v e s t i g a t i o n s . 5 THE STUDY AREA MATERIALS AND METHODS An i n t e n s i v e f i e l d s t u d y began a t the L i t t l e Campbel l R i v e r i n 1964 and c o n t i n u e d through 1965, S e v e r a l days were spent each week a t the s t u d y a r e a from March t h r o u g h September, b u t d u r i n g the w i n t e r v i s i t s averaged twice a month. Three l o c a l i t i e s were chosen f o r d e t a i l e d s t u d i e s : S t a t i o n D, a r e p r e s e n t a t i v e l e i u r u s h a b i t a t , S t a t i o n N, a r e p r e s e n t a t i v e t r a c h u r u s h a b i t a t , and S t a t i o n s L , M, and M ' , w h i c h i n c l u d e d the h y b r i d z o n e . C o l l e c t i o n n o t e s , e c o l o g i c a l d a t a , and o t h e r h a b i t a t c h a r a c t e r i s t i c s were r e c o r d e d . S a l i n i t i e s were r e c o r d e d w i t h a s e t o f t h r e e h y d r o -meters r e g i s t e r i n g d e n s i t i e s o f 1 .000-1 .011 , 1 .010-1 .021 , and 1 ,020-1 .031 s p e c i f i c g r a v i t y o f N a C l . Sometimes a B u c h l e r -C o t l o v e c h l o r i d o m e t e r was u s e d to determine s a l i n i t y near the e s t u a r y . These measurements were compared w i t h r e c o r d i n g s from the upper reaches o f the s t ream to d e t e c t s m a l l d i f f e r e n c e s i n s a l i n i t y t h a t might e x i s t as a r e s u l t o f t i d a l f l u x . T o t a l d i s s o l v e d s o l i d s were d e t e r m i n e d w i t h an R. C , c o n d u c t i v i t y b r i d g e . Water samples f o r pH measurements were c o l l e c t e d from the s t ream and i m m e d i a t e l y r e t u r n e d to the l a b o r a t o r y f o r d e t e r m i n a t i o n w i t h a Beckman pH m e t e r . Stream c u r r e n t s were measured w i t h a G u r l y c u r r e n t meter o r a t a b l e t e n n i s b a l l f l o a t e d on the s u r f a c e o v e r a known d i s t a n c e . The l a t t e r method was u s e d f o r m e a s u r i n g average c o n d i t i o n s over a l a r g e s e c t i o n o f the s t r e a m . 6 F i s h were c o l l e c t e d w i t h a common sense minnow s e i n e o f woven n y l o n w i t h 3 mm s t r e t c h e d mesh, The dimensions were 3m x 2m, and the l e a d l i n e was h e a v i l y w e i g h t e d to f a c i l i t a t e c o l l e c t i n g i n dense v e g e t a t i o n . Where s e i n i n g was i m p o s s i b l e , a v a r i e t y o f f i n e meshed d i p n e t s were u s e d . L i v e f i s h were t r a n s p o r t e d i n 8 g a l l o n s t y r o f o a m c o o l e r s , and c o l l e c t i o n s f o r m o r p h o l o g i c a l s t u d i e s were p r e s e r v e d i n 1 5 % b u f f e r e d f o r m a l i n . I n v e r t e b r a t e s were p r e s e r v e d i n 8 0 % e t h y l a l c o h o l . THE HABITAT A s m a l l c o a s t a l s t ream, the L i t t l e Campbel l R i v e r , was chosen f o r a d e t a i l e d s t u d y o f the s t i c k l e b a c k because i t i s r e a d i l y a c c e s s i b l e and because l e i u r u s and t r a c h u r u s are abundant t h e r e . I t i s l o c a t e d i n the extreme southwest c o r n e r o f B r i t i s h Columbia and empties i n t o Semiahmoo Bay a t White Rock ( F i g , 1 ) , The L i t t l e C a m b e l l R i v e r i s a p p r o x i m a t e l y 16% m i l e s l o n g . The w i d t h v a r i e s f rom l e s s than 10 f e e t i n the upper reaches to more than 3 6 f e e t a t the e s t u a r y . F o r most o f the l e n g t h i t i s e a s i l y waded, b u t i s o l a t e d p o o l s and s t r e t c h e s o f c h a n n e l s are as deep as seven f e e t . The water l e v e l f l u c t u a t e s c o n s i d e r a b l y . D u r i n g the s p r i n g i n t e r m i t t e n t f l o o d i n g o c c u r s so t h a t meadows a l o n g the banks are c o v e r e d w i t h t h i n s h e e t s of water , p a r t i c u l a r l y i n the upper r e a c h e s . On the o t h e r hand, the water l e v e l drops v e r y low i n summer and c u r r e n t may be s c a r c e l y n o t i c e a b l e . The narrow w i d t h and s h a l l o w waters are s u i t a b l e f o r e f f i c i e n t s e i n i n g and F i g u r e 1. L o c a t i o n of C o l l e c t i n g S t a t i o n s on the L i t t l e C a m p b e l l R i v e r . 8 f i e l d studies. Major habitats and t h e i r features are found i n Table I and Figure 2 shows the d i s t r i b u t i o n of sticklebacks i n the stream. The headwaters (Stations A-E) are approximately 7% miles long and lay at an a l t i t u d e of 220 feet. This habitat i s marshy with a mud bottom and dense aquatic vegetation that chokes sections of the stream i n the summer, Leiurus i s extremely abundant, and density estimates shown i n Table II convey some idea of t h e i r numbers. This race tends to congregate i n shallow pockets along the margins of the stream where vegetation i s most dense and i s less numerous where the banks are steep and vegetation i s sparse. Such a d i s t r i b u t i o n gives r i s e to marked l o c a l v a r i a t i o n s i n density (Table I I ) . In the spring males with breeding colours can be found a l l over the flooded meadows; ponds along the stream also have dense populations of l e i u r u s . The headwaters give way abruptly at Station E where the r i f f l e habitat (Stations E-K) begins, extending for the next 4 % miles. This section i s characterized by swift water, rocky bottom, lack of vegetation, and no sticklebacks. At Station K there i s again a rapid t r a n s i t i o n from the r i f f l e habitat to one more l i k e the headwaters, But there i s less vegetation, s l i g h t l y faster current, and some sand and s i l t , Leiurus i s less abundant here than i n the upper reaches (Table I I ) . A short distance downstream i s the hybrid zone, which w i l l be described i n a separate subsection. The lower reaches (Station N) occupy 3 / 4 mile and T a b l e I . A s y n o p s i s of h a b i t a t c h a r a c t e r i s t i c s i n the L i t t l e C a m p b e l l R i v e r . P h y s i c a l and c h e m i c a l data r e p r e s e n t average c o n d i t i o n s f r o m May t h r o u g h A u g u s t . HABITAT: Headwaters (leiurus habitat) STATION: (A-E) R i f f l e Section (E-K) Hybrid Zone (L-M1) , Lower Reaches (trachurus habitat) (N) Estuary (0-P) stream gradient water v e l o c i t y temperature s a l i n i t y t o t a l d i s s o l v e d s o l i d s PH water colour bottom vegetation vertebrates f i s h : 8 f t a mile 3 cm a second 16°C 0.189bo 65 ppm 6.8 medium tea colour; s l i g h t l y t u r b i d s o f t and muddy dense beds of Nuphar. Myosotis, Oenanthe, Glyceria. Carex. Typha, Po tamo ge ton; also Lemna and green algae Catostomus catostomus. Cottus asper. parr of Oncorhynchus kisutch. Salmo c l a r k i amphibians: Bufo boreas. Rana pretiosa. Hyla r e q i l l a . Ambystoma g r a c i l e invertebrates molluscs: Lymnaea. Physa. Ancylus. Musculium, Mar q a r i t i f e r a. Gyraulus crustaceans & annelids: amphipods and leeches abundent aquatic i n s e c t s : naiads of dragon and damselflies, S i a l i s , Nepa, Chironomidae, Chrysomelidae, Ephemoptera 32 f t a mile 74 cm a second 14°C 0.189bo 7.1 c l e a r rocky and rubble very sparse; some encrusting algae (Cladophora) C. asper. C. aleuticus. 0. kisutch. S. c l a r k i B. boreas Ancylus Trichoptera, Ephemeroptera, Plecoptera 12 f t a mile 23 cm a second 15°C 0 . 2 0 9 O O 118ppm 7.8 l i g h t tea colour to c l e a r ; s l i g h t l y t u r b i d intermingled with mud, medium grained sand, and some s i l t some Elodea and Myriophyllum; some Glyce r i a . Carex. Typha C. asper. C. a l e u t i c u s R. pr e t i o s a 11 f t a mile 10 cm a second 15°C 0.2l9bo 118ppm 7.8 co l o u r l e s s ; s l i g h t l y t u r b i d sand dense beds of Elodea C. asper. C. a l e u t i c u s . 0. kisutch, Platichthys s t e l l a t u s Musculium, Pisidium, M a r q a r i t i f e r a Gammaridae, Palaemonetes Trichoptera, Chrysomelidae, Chironomidae, Dixidae, naiads of damselflies 10 cm a second 16°C mixohaline (0.179bo to 1 2 9 b o ) t u r b i d mud, sand, s i l t , c l a y and some oyster beds dense beds of Ulva and some Enteromorpha C. asper. C. a l e u t i c u s . P. s t e l l a t u s . Oligocottus maculosus. Leptocottus armatus Crassostrea Balanus. Gammaridae, Palaemonetes % 100 50 • TRACHURUS O HYBRIDS • LEIURUS mm. A 0.2 P OA 0 F i g u r e 2 D i s t r i b u t i o n o f Campbel l R i v e r , f o r S t a t i o n was ' 1.5 7.8 2 2.8 3.5 N M' M L K DISTANCE FROM THE SEA IN MILES AND STATIONS races and h y b r i d s of s t i c k l e b a c k a t S t a t i o n s on the L i t t l e Data b a s e d on more than 6000 f i s h . M i n i m a l sample' s i z e 200, 16 A T a b l e I I , D e n s i t y e s t i m a t e s f o r l e i u r u s and t r a c h u r u s from s e v e r a l h a b i t a t s i n the L i t t l e C a m p b e l l R i v e r . Each f i g u r e r e p r e s e n t s the average c a t c h from t h r e e a d j a c e n t 15 f o o t s e i n e h a u l s . C o n s e c u t i v e f i g u r e s f o r a g i v e n date are s i m i l a r e s t i m a t e s made a t 200 f o o t i n t e r v a l s o f s t r e a m . D e n s i t i e s o f l e i u r u s i n 1965, n o t g i v e n , were l i k e those f o r 1964. L e i u r u s , 1964 S t a t i o n D S t a t i o n K S t a t i o n M March 6 March 13 A p r i l 5 A p r i l 23 1 .7 2.112 3 .97 1.138 2.101 3 .86 1.116 2.204 3 .9 1.27 2 .14 3.196 May 4 May 28 June 9 June 22 1.123 2.216 1.416 2.28 3.312 1.432 2.156 3.82 1.257 2 .46 3.315 March 16 A p r i l 18 May 3 June 12 1. 2 3, 1. 2. 3, 1, 2, 3, 1. 2. 3. 8 22 10 17 5 6 19 3 9 6 31 14 March 16 A p r i l 17 May 3 June 12 1 2, 3, 1 2, 3, 1, 2, 7 3 15 0 3 1 5 16 3 .8 1 .11 2 .1 3 .5 T r a c h u r u s 1964 S t a t i o n N S t a t i o n 0 1965 S t a t i o n N June 16 June 23 J u l y 6 J u l y 21 A u g u s t 8 1.212 1.9 June 8 1.43 J u l y 1 1.9 2.193 2.3 2 .21 2.5 3.247 3.23 1.184 1.2 June 18 1.12 J u l y 5 1 .6 2.202 2 .8 3 .7 2 .8 3 .16 2 .7 1.164 1.5 June 22 1.11 J u l y 14 1.12 2.221 2.3 2.20 2.3 3.202 3.5 3.8 1.175 1.26 June 26 1.19 J u l y 20 1.7 2.124 2 .7 2 .6 2.5 3.256 3.12 1.167 1.3 2 .206 2 .11 3.198 3.2 12 are 1% m i l e s f rom the e s t u a r y . The h a b i t a t has a sandy bot tom, a m i l d c u r r e n t , and dense beds o f E l o d e a w h i c h i s the o n l y a q u a t i c v e g e t a t i o n p r e s e n t , c o v e r i n g most o f the bottom i n the summer b u t d y i n g away i n the w i n t e r . T r a c h u r u s spawning grounds are l o c a t e d here a f t e r they m i g r a t e i n t o the s t ream i n l a t e s p r i n g to b r e e d . S a l i n i t y measurements made throughout the b r e e d i n g season show t h a t the water i s f r e s h and t h a t i t does not d i f f e r much from the s a l i n i t y o f water i n the upper reaches (Table I ) . T a b l e I I i n d i c a t e s the l a r g e numbers o f t r a c h u r u s t h a t b r e e d i n t h i s h a b i t a t . The d a t a a l s o demonstrate a s t r i k i n g decrease i n numbers f o u n d i n 1965. T r a c h u r u s spawning grounds are c o n c e n t r a t e d i n t o a s h o r t s e c t i o n o f the s t ream and t h i s h a b i t a t was e x t e n s i v e l y s e i n e d i n 1964. The h e a v i l y w e i g h t e d l e a d l i n e o f the s e i n e p r o b a b l y d e s t r o y e d many n e s t s and f r y o f t r a c h u r u s . The decrease i n d e n s i t y f o u n d i n 1965 s u g g e s t s t h a t t r a c h u r u s homes to a g i v e n s t ream to spawn s i n c e many o f the f r y t h a t would have r e t u r n e d to the s t ream i n 1965 might have been k i l l e d i n 1964. T r a c h u r u s i s c l o s e l y a s s o c i a t e d w i t h E l o d e a , and s i n c e E l o d e a i s u n i f o r m l y d i s t r i b u t e d t h i s p r o b a b l y a c c o u n t s f o r the more u n i f o r m numbers a l o n g the s tream when t h a t r a c e i s compared w i t h l e i u r u s (Table I I ) . The e s t u a r y ( S t a t i o n s 0-P) i s an u n s t a b l e h a b i t a t w i t h l a r g e f l u c t u a t i o n s i n water l e v e l , m i x o h a l i n e water , and dense beds o f U l v a . S m a l l numbers o f t r a c h u r u s can be f o u n d t h r o u g h o u t the summer (Table II)„ 13 THE HYBRID ZONE T h i s s e c t i o n o f the s t ream i s one m i l e l o n g , and T a b l e I shows t h a t f o r f e a t u r e s such as bot tom t y p e , v e g e t a t i o n , c u r r e n t , and water q u a l i t y , i t i s i n t e r m e d i a t e between the headwaters h a b i t a t and the lower r e a c h e s . The h y b r i d zone ( S t a t i o n s L - M 1 ) i s s e p e r a t e d from the t r a c h u r u s spawning grounds by a % m i l e s t r e t c h o f c l a y and s i l t bottom t h a t i s d e v o i d o f v e g e t a t i o n and s t i c k l e b a c k s . V e r y few t r a c h u r u s m i g r a t e upstream beyond t h i s r e g i o n and, c o n v e r s e l y , v e r y few l e i u r u s are f o u n d downstream below i t ( F i g u r e 2 ) , M i x e d b r e e d i n g p o p u l a t i o n s o f l e i u r u s and t r a c h u r u s do not o c c u r i n the h y b r i d z o n e . The numbers o f l e i u r u s are much s m a l l e r when compared w i t h numbers i n the headwaters (Table II) and o n l y o c c a s i o n a l i n d i v i d u a l s o f t r a c h u r u s are c a p t u r e d , b u t a t S t a t i o n s M and M'^where the l a r g e s t numbers o f l e i u r u s and t r a c h u r u s are caught t o g e t h e r h y b r i d swarms have d e v e l o p e d . The r a t i o o f l e i u r u s , h y b r i d s , and t r a c h u r u s , i s 1 0 J 8 : 1 . A t S t a t i o n L numbers o f l e i u r u s i n c r e a s e and d e n s i t y i s l i k e t h a t shown f o r S t a t i o n K, b u t numbers o f h y b r i d s and o f t r a c h u r u s decrease ( F i g u r e 2 ) . H y b r i d s are a l s o common i n a s m a l l pond a d j a c e n t to the s t ream a t S t a t i o n M (Pond M i n F i g u r e 1 ) . The o u t s t a n d i n g f e a t u r e s o f the h y b r i d zone are i t s narrowness , i t s s h a r p l y d e f i n e d b o u n d a r i e s , and i t s i n t e r m e d i a t e p r o p e r t i e s between the headwaters ( l e i u r u s h a b i t a t ) and the lower reaches ( t r a c h u r u s h a b i t a t ) . The F]_ and b o t h b a c k c r o s s c l a s s e s of h y b r i d s may be i d e n t i f i e d by t h e i r morphology as w i l l be demonstra ted l a t e r . 14 If the hybrid zone i s divided i n hal f between Stations M and M1 the numbers of f i s h i n the upper and lower h a l f of the hybrid zone are, respectivelys for leiurus backcrosses 132 and 38, for trachurus backcrosses 41 and 76, When the data se tested with a contingency table a value of P < ,005 i s obtained i n d i c a t i n g that hybrids are not randomly d i s t r i b u t e d , Backcrosses to trachurus are mostly i n the lower h a l f of the hybrid zone nearest Station N, and backcrosses to leiurus are mostly i n the upper h a l f nearest Station K, 15 MORPHOLOGICAL ANALYSIS MATERIALS AND METHODS L e i u r u s and t r a c h u r u s d i f f e r i n a number o f m o r p h o l o g i c a l c h a r a c t e r s . Those chosen f o r a n a l y s i s were number o f l a t e r a l p l a t e s , number o f g i l l r a k e r s , e l e c t r o p h o r e t i c p a t t e r n s , s t a n d a r d l e n g t h a t s e x u a l m a t u r i t y , body depth i n t o s t a n d a r d l e n g t h a t s e x u a l m a t u r i t y , and number o f d o r s a l and a n a l r a y s . These c h a r a c t e r s were s e l e c t e d because they c o u l d be most e a s i l y and o b j e c t i v e l y measured, and because the two r a c e s showed c o n s p i c u o u s d i f f e r e n c e s i n those c h a r a c t e r s . Other d i f f e r e n c e s f o r example, number o f v e r t e b r a e , l e n g t h and shape o f d o r s a l and p e l v i c s p i n e s , , and c o l o u r p a t t e r n s , were not u s e d . Procedures f o r counts and measurements were i n most i n s t a n c e s those s u g g e s t e d b y Hubbs and L a g l e r ( 1 9 5 8 ) „ The e x c e p t i o n s were counts o f l a t e r a l bony p l a t e s and e l e c t r o p h o r e t i c a n a l y s i s . A l l l a t e r a l p l a t e s were c o u n t e d i n c l u d i n g the s m a l l a n t e r i o r ones and those i n the c a u d a l k e e l . Counts were made u s i n g a b i n o c u l a r d i s s e c t i n g scope and w i t h the a i d o f a p r o b e . F i s h s m a l l e r than about 3 cm were s t a i n e d w i t h a few c r y s t a l s of a l i z a r i n dye i n 2% KOH f o r 24 h o u r s b e f o r e p l a t e s were c o u n t e d to r e n d e r the s m a l l a n t e r i o r and p o s t e r i o r p l a t e s c o n s p i c u o u s . D r . H . T s u y u k i a t the F i s h e r i e s R e s e a r c h Board T e c h n o l o g i c a l S t a t i o n , Vancouver , d i d the e l e c t r o p h o r e t i c a n a l y s i s on muscle p r o t e i n u s i n g p r e v i o u s l y p u b l i s h e d methods ( T s u y u k i , 1 9 6 2 ) „ O n l y s e x u a l l y mature f i s h were u s e d to measure s t a n d a r d l e n g t h and body depth i n t o s t a n d a r d l e n g t h so t h a t a more u n i f o r m 16 c o m p a r i s o n among the r a c e s and h y b r i d s c o u l d be made. V e r n i e r c a l l i p e r s were used to make a l l measurements. S t i c k l e b a c k s u s e d i n the m o r p h o l o g i c a l a n a l y s i s were c o l l e c t e d from the L i t t l e Campbel l R i v e r d u r i n g the s p r i n g and summer o f 1964 and 1965. In o r d e r to c i r c u m s c r i b e and i d e n t i f y the morphology o f each race samples were c o l l e c t e d from a l l o p a t r i c p o p u l a t i o n s o f l e i u r u s and t r a c h u r u s . These samples a s s u r e , as much as p o s s i b l e , a g a i n s t the e f f e c t s o f h y b r i d i z a t i o n and i n t r o g r e s s i o n between the r a c e s . F u r t h e r m o r e , they s e r v e as c o n t r o l s so t h a t once t h e i r m o r p h o l o g i c a l c h a r a c t e r i s t i c s are known the samples can be compared w i t h s y m p a t r i c p o p u l a t i o n s o f l e i u r u s and t r a c h u r u s . In t h i s way the e f f e c t s and e x t e n t o f h y b r i d i z a t i o n and i n t r o g r e s s i o n can be d e t e r m i n e d . Samples from a l l o p a t r i c p o p u l a t i o n s o f l e i u r u s were t a k e n from the headwaters a t S t a t i o n s A , B, and D . I t may be r e a s o n a b l y assumed t h a t these p o p u l a t i o n s are a l l o p a t r i c . Thousands o f i n d i v i d u a l s have been examined i n the headwaters , b u t not one t r a c h u r u s o r h y b r i d was f o u n d . C o l l e c t i o n s from a l l o p a t r i c p o p u l a t i o n s o f t r a c h u r u s were made a t S t a t i o n P i n the e s t u a r y , and s i n c e n e i t h e r l e i u r u s nor h y b r i d s were f o u n d t h e r e ( F i g u r e 2) i t may a g a i n be assumed t h a t these p o p u l a t i o n s are a l l o p a t r i c . I n d i v i d u a l s o f each r a c e were f o u n d t o g e t h e r a t S t a t i o n s L , M, and M' d u r i n g the b r e e d i n g season t o g e t h e r w i t h numerous v a r i a b l e and i n t e r m e d i a t e i n d i v i d u a l s . Samples from t h i s s e c t i o n o f the s t ream - c a l l e d the h y b r i d zone - were u s e d 17 to determine the presence o f h y b r i d i z a t i o n . H y b r i d s i n f i s h are a l m o s t always i n t e r m e d i a t e between the p a r e n t a l types i n t h e i r m o r p h o l o g i c a l f e a t u r e s (Hubbs, 1955), and the presence o f such i n d i v i d u a l s t o g e t h e r w i t h b o t h p a r e n t a l types p r o v i d e s s t r o n g c i r c u m s t a n t i a l e v i d e n c e f o r h y b r i d i z a t i o n . M o r e o v e r , F^ h y b r i d s u s u a l l y show s t r i c t i n t e r m e d i a c y between the p a r e n t s so t h a t the presence o f i n d i v i d u a l s t h a t approach one or the o t h e r p a r e n t more c l o s e l y i n t h e i r morphology i n d i c a t e s b a c k c r o s s i n g has o c c u r r e d (Hubbs, 1955), Thus , once the morphology f o r the c h a r a c t e r s i n d i c a t e d had been d e t e r m i n e d i n a l l o p a t r i c p o p u l a t i o n s , a c o m p a r i s o n w i t h samples from the zone o f o v e r l a p between l e i u r u s and t r a c h u r u s may i n d i c a t e the presence o f h y b r i d i z a t i o n and b a c k c r o s s i n g , A p r e l i m i n a r y a n a l y s i s o f p l a t e counts (which are most r e l i a b l e f o r i d e n t i f y i n g h y b r i d s as w i l l be shown) demonstra ted t h a t the b a c k c r o s s c l a s s e s were nonrandomly d i s t r i b u t e d . Knowing t h i s c a r e was taken to sample the h y b r i d zone u n i f o r m l y be s e i n i n g f o r f i s h o v e r the e n t i r e d i s t a n c e from S t a t i o n L to S t a t i o n M ' . To c o n f i r m the presence o f h y b r i d s caught i n w i l d samples o f each r a c e , a r t i f i c a l l y produced r e c i p r o c a l F^ h y b r i d s and b a c k c r o s s e s were r e a r e d under u n i f o r m c o n d i t i o n s i n the l a b o r a t o r y . Parents u s e d to o b t a i n l e i u r u s and t r a c h u r u s o f f s p r i n g were c o l l e c t e d from the same a l l o p a t r i c p o p u l a t i o n s ment ioned e a r l i e r . R e c i p r o c a l F^ h y b r i d s were p r o d u c e d by c r o s s i n g p a r e n t s o f each r a c e , a g a i n c o l l e c t e d from a l l o p a t r i c p o p u l a t i o n s , i n b o t h c o m b i n a t i o n s , B a c k c r o s s e s were made by 18 f i r s t c o l l e c t i n g presumed h y b r i d s f rom the h y b r i d z o n e . These i n d i v i d u a l s were i d e n t i f i e d by t h e i r p l a t e c o u n t s , and o n l y i n d i v i d u a l s t h a t were s t r i c t l y i n t e r m e d i a t e between the r a c e s were u s e d . One can never be c e r t a i n t h a t these i n d i v i d u a l s are F^ h y b r i d s , b u t e v i d e n c e p r e s e n t e d i n a n o t h e r s e c t i o n (Seasonal I s o l a t i o n ) shows t h a t the sexes o f h y b r i d s are l a r g e l y p r e v e n t e d from i n t e r b r e e d i n g by s e a s o n a l i s o l a t i o n . The p o s s i b i l i t y o f an i n t e r m e d i a t e h y b r i d b e i n g an F2 i s t h e r e f o r e u n l i k e l y . B a c k c r o s s e s were then made by c r o s s i n g presumed F^ h y b r i d s w i t h p a r e n t s o f each race i n the f o u r r e c i p r o c a l c o m b i n a t i o n s . F e r t i l i z e d eggs and o f f s p r i n g were r e a r e d i n d e c h l o r i n a t e d f r e s h w a t e r a t the Vancouver P u b l i c A q u a r i u m , Methods o f t a k i n g and f e r t i l i z i n g eggs , i n c u b a t i n g eggs , and r e a r i n g o f f s p r i n g are f o u n d i n the s e c t i o n on P o s t m a t i n g I s o l a t i n g Mechanisms. Eggs were h e l d a t two t e m p e r a t u r e s , 1 4 ° C and 1 8 ° C , c o n t r o l l e d w i t h t h e r m o s t a t s suspended i n water b a t h s . F r y were r e a r e d a t room t e m p e r a t u r e , which v a r i e d from 1 7 ° C to 2 2 ° C , The o f f s p r i n g were between 17 and 18 weeks o l d when they were p r e s e r v e d f o r m o r p h o l o g i c a l a n a l y s i s , and a t t h a t age they were between 2.8 and 3,7 cm l o n g . The same c h a r a c t e r s were a n a l y z e d on r e a r e d and on w i l d f i s h . ALLOPATRIC POPULATIONS L a t e r a l P l a t e C o u n t s . L e i u r u s and t r a c h u r u s are e a s i l y i d e n t i f i e d b y number o f p l a t e s ( F i g u r e 3) s i n c e they are w i d e l y d i v e r g e n t w i t h no o v e r l a p i n c o u n t s . Counts o f 3 % 30 20 10 0 40 30 20 10 0 40 30 20 10 0 TRACHURUS N=890 X=32.7 30 31 32 33 34 35 HYBRIDS N-493 X=17 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 LEIURUS 4 5 6 7 L a t e r a l p l a t e counts f o r a l l o p a t r i c p o p u l a t i o n s o f l e i u r u s and t r a c h u r u s and o f h y b r i d s . N - sample s i z e ; X - mean. 20 to 7 are d i a g n o s t i c f o r l e i u r u s , and 30 to 35 f o r t r a c h u r u s . G i l l Raker C o u n t s . T h i s c h a r a c t e r p r o v i d e s a u s e f u l means o f d i s t i n g u i s h i n g r a c e s ( F i g u r e 4 ) , and i t can be seen t h a t l e i u r u s averages c o n s i d e r a b l y fewer r a k e r s than t r a c h u r u s . There i s an o v e r l a p o f 4% i n c o u n t s , which i s d e t e r m i n e d by e s t i m a t i n g the p e r c e n t o f i n d i v i d u a l s i n t h a t r a c e w i t h s m a l l e s t numbers o f i n d i v i d u a l s i n the o v e r l a p p i n g z o n e . E l e c t r o p h o r e s i s . L i k e p l a t e s t h i s c h a r a c t e r can be u s e d to i d e n t i f y each r a c e because t h e r e are bands t h a t are unique to each ( F i g u r e 5 ) „ S t a n d a r d Length o f A d u l t s . P o p u l a t i o n s o f l e i u r u s and t r a c h u r u s may be d i f f e r e n t i a t e d b y a d u l t l e n g t h ( n u p t i a l l y c o l o u r e d males and g r a v i d females ) l e i u r u s h a v i n g a s m a l l e r average s i z e than t r a c h u r u s . However, an o v e r l a p of 50% makes t h i s c h a r a c t e r l e s s u s e f u l than o t h e r s ( F i g u r e 6 ) . Body Depth i n t o S t a n d a r d Length o f A d u l t s . L e i u r u s has a more chunky body and t r a c h u r u s a more t e r e t e body ( F i g u r e 6 ) . F o r t h i s c h a r a c t e r t h e r e i s an o v e r l a p o f 14% between r a c e s . D o r s a l and A n a l Rays . The r a c e s d i f f e r i n d o r s a l and a n a l r a y c o u n t s ( F i g u r e 6) and i n each c h a r a c t e r l e i u r u s averages fewer r a y s than does t r a c h u r u s . O v e r l a p i n d o r s a l r a y s i s 36% and i n a n a l r a y s i s 31%. THE HYBRID ZONE Data f o r the m o r p h o l o g i c a l a n a l y s i s o f h y b r i d s are p r e s e n t e d a l o n g w i t h those f o r a l l o p a t r i c p o p u l a t i o n s so t h a t comparisons can be more e a s i l y made. A l t h o u g h b o t h r a c e s are p r e s e n t i n the h y b r i d zone ( F i g u r e 2) o n l y the h y b r i d s are i n c l u d e d i n t h i s a n a l y s i s . H y b r i d s were i d e n t i f i e d by t h e i r 12 13 14 15 16 17 18 19 20 21 22 23 24 25 GILL RAKER COUNTS. Figure 4 . G i l l raker counts for a l l o p a t r i c populations of leiurus and trachurus and of hybrids. N - sample size; X - mean. T r a c h u r u s F i g u r e 5 . E l e c t r o p h o r e t i c b a n d i n g p a t t e r n s f o r a l l o p a t r i c p o p u l a t i o n s o f l e i u r u s and t r a c h u r u s and o f h y b r i d s . D i a g n o s t i c bands o f each race are i n d i c a t e d by the a r r o w s . Sample s i z e s are 60 l e i u r u s ? 60 t r a c h u r u s ? 120 h y b r i d s ; w i t h i n the h y b r i d c l a s s i n d i v i d u a l s had e i t h e r b o t h bands d i a g n o s t i c f o r each r a c e p r e s e n t or one band d i a g n o s t i c f o r one o r the o t h e r p a r e n t . These may be i n t e r p r e t e d as e i t h e r F^ h y b r i d s o r e i t h e r b a c k c r o s s c l a s s . Percentages f o r the t h r e e c l a s s e s w e r e : 11.8% F±; 80.2% b a c k c r o s s e s to l e i u r u s ? 8% b a c k c r o s s e s to t r a c h u r u s . 23 DORSAL RAY COUNTS ANAL RAY COUNTS Figure 6. Morphological c h a r a c t e r i s t i c s for a l l o p a t r i c of leiurus and trachurus and of hybrids. N - sample size; X - mean. populations 24 p l a t e c o u n t s , w h i c h i n c l u d e s a l l s t i c k l e b a c k s w i t h c o u n t s i n t e r m e d i a t e to counts o b t a i n e d f o r a l l o p a t r i c p o p u l a t i o n s o f each r a c e ( i . e . w i t h counts o v e r 7 and under 3 0 ) . However, when samples were c o l l e c t e d to p r o v i d e m a t e r i a l f o r c o n s t r u c t i n g a h y b r i d i n d e x a l l s t i c k l e b a c k s taken i n the h y b r i d zone were i n c l u d e d (see The H y b r i d I n d e x ) . L a t e r a l P l a t e C o u n t s . H y b r i d s show a complete range o f p l a t e c o u n t s i n t e r m e d i a t e between the p a r e n t a l types ( F i g u r e 3 ) . Such a l a r g e amount o f v a r i a t i o n f o r a c h a r a c t e r so d i v e r g e n t i n the r a c e s s u g g e s t s t h a t b a c k c r o s s i n g takes p l a c e . The predominance o f counts a p p r o a c h i n g the l e i u r u s p a r e n t f u r t h e r s u g g e s t s t h a t the m a j o r i t y o f b a c k c r o s s e s are to t h a t r a c e . I n s p e c t i o n o f d a t a f o r o t h e r m o r p h o l o g i c a l f e a t u r e s o f h y b r i d s show a p a t t e r n s i m i l a r to t h a t o b t a i n e d from p l a t e c o u n t s ( F i g u r e s 4, 5, and 6) s i n c e h y b r i d s are always more or l e s s i n t e r m e d i a t e i n c o u n t s and measurements. F r e q u e n c y d i s t r i b u t i o n s f o r a l l c h a r a c t e r s b u t d o r s a l r a y s are skewed towards the l e i u r u s p a r e n t s u g g e s t i n g c o n s i d e r a b l e b a c k c r o s s i n g to t h a t p a r e n t ( f o r p e r c e n t a g e s o f h y b r i d c l a s s e s o b t a i n e d from e l e c t r o p h o r e t i c a n a l y s i s see F i g u r e 5 ) . THE HYBRID INDEX Once the presence o f h y b r i d s i s demonstra ted the p o s s i b i l i t y o f i n t r o g r e s s i o n e x i s t s . I n t r o g r e s s i o n can have p r o f o u n d and f a r r e a c h i n g e f f e c t s i n the e v o l u t i o n o f s p e c i e s (Anderson, 1949; S t e b b i n s , 1959) making a c r i t i c a l e x a m i n a t i o n n e c e s s a r y . I f one a t tempt to e s t a b l i s h i n t r o g r e s s i o n 25 o r i t s d i r e c t i o n , to one o r the o t h e r p a r e n t , u s i n g any s i n g l e c h a r a c t e r the r e s u l t s may be v e r y m i s l e a d i n g f o r a number o f r e a s o n s . One needs o n l y to c o n s i d e r the g e n e t i c b a s i s -dominance, o p p o s i t i o n a l e f f e c t s , and o t h e r i n t e r a c t i o n s o f genotypes - to r e a l i z e t h i s . More o f t e n e f f e c t s o f r e c o m b i n a t i o n make the use of one c h a r a c t e r l e s s r e l i a b l e . F o r example, p l a t e s can be r e l i a b l y u s e d to i d e n t i f y a h y b r i d b u t i t was f o u n d t h a t a few h y b r i d s cannot be r e c o g n i z e d u s i n g p l a t e s a l o n e . B a c k c r o s s i n g and r e c o m b i n a t i o n y i e l d s some o f f s p r i n g w i t h c o u n t s l i k e those o f the p a r e n t race so they cannot be i d e n t i f i e d w i t h t h a t c h a r a c t e r , b u t r a k e r counts r e v e a l t h e i r h y b r i d o r i g i n . And so i t i s f o r any s i n g l e c h a r a c t e r so t h a t c o m b i n i n g a number of", c h a r a c t e r s i n t o a s i n g l e i n d e x and w e i g h i n g each c h a r a c t e r r e l a t i v e to i t s u s e f u l n e s s p r o v i d e s a more p o w e r f u l t o o l to d e t e c t i n t r o g r e s s i o n and i t s d i r e c t i o n . F r e s h samples o f 40 l e i u r u s and 40 t r a c h u r u s were c o l l e c t e d from a l l o p a t r i c p o p u l a t i o n s a l o n g w i t h 144 i n d i v i d u a l s taken t h r o u g h o u t the h y b r i d z o n e . These were r e t u r n e d to the l a b o r a t o r y a l i v e , and once counts and measurements were made the f i s h were i m m e d i a t e l y f r o z e n i n p r e p a r a t i o n f o r e l e c t r o p h o r e t i c a n a l y s i s o f muscle p r o t e i n . Each i n d i v i d u a l was a n a l y z e d f o r f i v e m o r p h o l o g i c a l f e a t u r e s . D o r s a l and a n a l r a y counts were e x c l u d e d from the i n d e x because they were f o u n d u s e l e s s (see Morphology o f L a b o r a t o r y Reared F i s h ) . : The method o f A n d e r s o n (1956) f o r c o n s t r u c t i n g a h y b r i d 26 i n d e x p r o v e d s a t i s f a c t o r y i n t h i s a n a l y s i s . The f i v e c h a r a c t e r s u s e d i n the i n d e x were i n c l u d e d because of t h e i r demonstra ted s u c c e s s i n i d e n t i f y i n g the r a c e s and h y b r i d s (see above and Morphology o f L a b o r a t o r y Reared F i s h ) , Methods f o r s c o r i n g and w e i g h t i n g c h a r a c t e r s are p r e s e n t e d below, but. i t may be u s e f u l to r e v i e w b r i e f l y the u t i l i t y o f a h y b r i d i n d e x . The r a t i o n a l e b e h i n d s c o r i n g and w e i g h t i n g o f c h a r a c t e r s i s the f o l l o w i n g . Any c h a r a c t e r t h a t i s d i s c r e t e and w i d e l y d i v e r g e n t between r a c e s i s g i v e n more s c o r e s than a l e s s d i v e r g e n t c h a r a c t e r . T h i s i s so because t h e r e can be a w i d e r spect rum o f v a r i a t i o n e x p r e s s e d i n the h y b r i d c l a s s e s a l l o w i n g i n t e r m e d i a t e c o u n t s o r measurements (F-|_ h y b r i d s ) and counts o r measurements more o r l e s s c l o s e l y a p p r o a c h i n g one or the o t h e r p a r e n t ( b a c k c r o s s i n g o r i n t r o g r e s s i o n ) to be r e a l i z e d . I f the r a c e s are l e s s d i v e r g e n t not so much v a r i a t i o n i s e x p r e s s e d i n h y b r i d s , and c o n s e q u e n t l y fewer s c o r e s can be u s e d . F i n a l l y i f much o v e r l a p e x i s t s i n a c h a r a c t e r o n l y t h r e e s c o r e s can be a s s i g n e d , one f o r each r a c e and one f o r h y b r i d s . Where l i t t l e o v e r l a p i s p r e s e n t o n l y the extreme counts o f each r a c e are grouped f o r a s c o r e p r o v i d i n g f o r a more e f f i c i e n t s e p a r a t i o n o f r a c e s f r o m h y b r i d s , and o f r a c e s from one a n o t h e r , Weights are a s s i g n e d i n p r o p o r t i o n to the u s e f u l n e s s o f a c h a r a c t e r . I f the c h a r a c t e r i s o b j e c t i v e l y d e t e r m i n e d , d i a g n o s t i c f o r races and h y b r i d s , and w i t h no o r l i t t l e o v e r l a p , i t i s a s s i g n e d a maximum w e i g h t . As these c r i t e r i a d i m i n i s h or o v e r l a p i n c r e a s e s the c h a r a c t e r i s w e i g h t e d l e s s . 27 A f t e r a l l c h a r a c t e r s have been a n a l y z e d f o r an i n d i v i d u a l and the w e i g h t s f o r the f i v e c h a r a c t e r s d e t e r m i n e d they are added t o g e t h e r to g i v e an i n d e x v a l u e . In t h i s way i n d e x v a l u e s are o b t a i n e d f o r a l l i n d i v i d u a l s . Then l i k e i n d e x v a l u e s are grouped and p r e s e n t e d i n a f r e q u e n c y d i s t r i b u t i o n . T h i s procedure i s c a r r i e d out i n d e p e n d e n t l y f o r a l l o p a t r i c and s y m p a t r i c p o p u l a t i o n s so t h a t the two can be compared and thus the e x i s t e n c e and d i r e c t i o n o f b a c k c r o s s i n g o r i n t r o g r e s s i o n e v a l u a t e d . F i n a l l y , i t s h o u l d be r e a l i z e d t h a t one r a c e ( l e i u r u s ) always r e c e i v e s m i n i m a l w e i g h t s o f z e r o , and the o t h e r race always r e c e i v e s maximal w e i g h t s f o r each c h a r a c t e r . S c o r i n g and W e i g h t i n g  L a t e r a l P l a t e s . T h i s was g i v e n seven s c o r e s , one each f o r the f o l l o w i n g groups o f p l a t e c o u n t s ; 3-5 ( l e i u r u s ) , 30-35 ( t r a c h u r u s ) . 17-20 ( i n t e r m e d i a t e ) , 8-12 and 13-16 (approach-i n g l e i u r u s ) , 21-24 and 25-29 ( a p p r o a c h i n g t r a c h u r u s ) . The maximum w e i g h t o f 12 was a s s i g n e d to p l a t e s because i t i s o b j e c t i v e l y measured, as a l l c h a r a c t e r s u s e d a r e , i t i s d i a g n o s t i c o f r a c e s and h y b r i d s and i s w i d e l y d i v e r g e n t w i t h no o v e r l a p . G i l l R a k e r s . F i v e s c o r e s were a s s i g n e d to raker-c o u n t s t 12-14 ( l e i u r u s ) , 23-25 ( t r a c h u r u s ) . 18-19 ( i n t e r m e d i a t e ) , 15-17 ( a p p r o a c h i n g l e i u r u s ) , 20-22 ( a p p r o a c h i n g t r a c h u r u s ) . F o r t h i s c h a r a c t e r and the r e m a i n i n g ones , e x c e p t i n g e l e c t r o -p h o r e t i c p a t t e r n s , o n l y the extreme counts o f l e i u r u s and t r a c h u r u s 28 were grouped f o r reasons d i s c u s s e d a b o v e . Rakers f u l f i l l the c r i t e r i a f o r maximal w e i g h t i n g b u t f o r a s m a l l o v e r l a p o f 4%. A c c o r d i n g l y they were w e i g h t e d l e s s than p l a t e s , w i t h a v a l u e o f 10. E l e c t r o p h o r e t i c P a t t e r n s , O n l y t h r e e s c o r e s c o u l d be a s s i g n e d t h i s c h a r a c t e r s i n c e d i v e r g e n c e between races i s s m a l l . The one band d i a g n o s t i c to l e i u r u s r e c e i v e d a s c o r e , the o t h e r d i a g n o s t i c to t r a c h u r u s r e c e i v e d a s e c o n d , and i n d i v i d u a l s w i t h b o t h p r e s e n t r e c e i v e d a t h i r d . However, b a n d i n g p a t t e r n s f u l f i l l the r e m a i n i n g c r i t e r i a and so were w e i g h t e d 10, f o r a l t h o u g h they a re l e s s d i v e r g e n t than r a k e r s t h e r e i s no o v e r l a p . S t a n d a r d Length o f A d u l t s . S i z e s were d i v i d e d i n t o o n l y t h r e e c l a s s e s because o f the 50% o v e r l a p between r a c e s . Scores w e r e . 2 . 8 - 3 . 5 ( l e i u r u s ) . 5 . 4 - 6 , 3 ( t r a c h u r u s ) , A 2 . 9 - 5 . 3 ( i n t e r m e d i a t e s ) . A w e i g h t o f 4 was a s s i g n e d to s i z e because o f c o n s i d e r a b l e o v e r l a p . Body Depth i n t o S t a n d a r d L e n g t h , Scores were as f o l l o w s ? 3 , 1 - 3 . 5 ( l e i u r u s ) . 4 . 5 - 4 , 9 ( t r a c h u r u s ) , 3 , 6 - 4 , 4 ( i n t e r m e d i a t e s ) , The c h a r a c t e r was w e i g h t e d 6, The 14% o v e r l a p w a r r a n t s a h e a v i e r w e i g h t than s i z e b u t l e s s than b a n d i n g p a t t e r n s o r r a k e r s . Weights f o r the f i v e c h a r a c t e r s were summed f o r e v e r y i n d i v i d u a l , T r a c h u r u s always r e c e i v e d maximum w e i g h t s so t h a t the h i g h e s t i n d e x p o s s i b l e was 12 + 10 + 10 + 6 + 4 = 42 . L e i u r u s r e c e i v e d minimum v a l u e s f o r a l l c h a r a c t e r s so the l o w e s t i n d e x t h a t c o u l d be o b t a i n e d was z e r o . More or 2 9 l e s s i n t e r m e d i a t e i n d i v i d u a l s r e c e i v e d more o r l e s s i n t e r m e d i a t e i n d i c e s , I n d i c e s o f l e i u r u s and t r a c h u r u s from a l l o p a t r i c p o p u l a t i o n s are d i s t i n c t and w e l l s e p a r a t e d ( F i g u r e 7 ) „ However, the i n t e g r i t y o f the r a c e s b r e a k s down i n the h y b r i d z o n e . They are no l o n g e r s e p a r a t e d by a w e l l d e f i n e d gap, and the m o r p h o l o g i c a l f e a t u r e s o f each race become b l u r r e d w i t h one a n o t h e r . I t i s a l s o o b v i o u s t h a t l e i u r u s i s more a f f e c t e d t h a n t r a c h u r u s , presumably as a r e s u l t o f gene f l o w . The absence o f a d i s t i n c t i n t e r m e d i a t e mode s u g g e s t s t h a t F^ h y b r i d s are r e l a t i v e l y few, and the s c a r c i t y o f i n d i v i d u a l s a p p r o a c h i n g t r a c h u r u s i n d i c a t e s t h a t b a c k c r o s s e s to t h a t r a c e are r a r e . R e s u l t s f rom l a b o r a t o r y r e a r e d f i s h , p r e s e n t e d next , f u r t h e r c o n f i r m these i n t e r p r e t a t i o n s , LABORATORY REARED FISH L a t e r a l P l a t e C o u n t s , I t i s c l e a r f rom l a b o r a t o r y r e a r e d f i s h t h a t numbers o f p l a t e s are g e n e t i c a l l y c o n t r o l l e d ( F i g u r e 8 ) „ A l t h o u g h they were r e a r e d u n i f o r m l y the numbers of p l a t e s i n o f f s p r i n g o f l e i u r u s and t r a c h u r u s are d i s t i n c t and s e p e r a t e d b y a wide g a p . F u r t h e r m o r e , the range o f v a r i a t i o n f o r p l a t e s o f l a b o r a t o r y r e a r e d f i s h i s the same as t h a t f o r w i l d caught f i s h , e x c e p t t h a t no l a b o r a t o r y r e a r e d t r a c h u r u s h a d c o u n t s as low as 30 (nor d i d t h e i r p a r e n t s ) , The F^ h y b r i d s are l a r g e l y i n t e r m e d i a t e . But a s l i g h t s h i f t towards t r a c h u r u s s u g g e s t s some dominance by t h a t p a r e n t . The range o f v a r i a t i o n e x p r e s s e d i n Fj_ h y b r i d s accounts f o r o n l y a p a r t o f the v a r i a b i l i t y e n c o u n t e r e d i n w i l d h y b r i d s , w h i c h 20 HYBRID INDEX VALUES 20 22 42 15 10 S O U J 1 10 15 20L F i g u r e JUT LEIURUS TRACHURUS F r e q u e n c y d i s t r i b u t i o n o f the h y b r i d i n d e x . I n v e r t e d h i s t o g r a m s r e p r e s e n t a l l o p a t r i c p o p u l a t i o n s ? u p r i g h t h i s t o g r a m s r e p r e s e n t samples f rom the h y b r i d o z o n e . 3 1 I l I l I i I I i i l l I I i i i i i I i l i i i i i i i i i i I 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 PLATE COUNTS Figure 8. Lateral plate counts for laboratory reared (black histograms) and of wild caught f i s h (hatched histograms). Leiurus used as parents to obtain o f f s p r i n g had plate counts from 3-5; trachurus counts 32-34, Data based on 6 i n t r a r a c i a l crosses with leiurus parents, 6 i n t r a -r a c i a l crosses with trachurus parents, 7 i n t e r r a c i a l crosses for F^ hybrids, and 7 backcrosses. Approximately equal numbers of reci p r o c a l crosses made. 32 f u r t h e r c o n f i r m s the presence o f b a c k c r o s s i n g i n the h y b r i d z o n e , B a c k c r o s s progeny r e c o v e r e d from presumed c r o s s e s to l e i u r u s have p l a t e counts from 4-17. Of the t o t a l , 66% o f the o f f s p r i n g are i n t e r m e d i a t e i n c o u n t s between F-^'s and l e i u r u s , and can t h e r e f o r e be r e c o g n i z e d as b a c k c r o s s e s u s i n g p l a t e counts a l o n e . But 29% o f the progeny have counts f rom 4-7, and they would be i d e n t i f i e d as l e i u r u s i f p l a t e s were u s e d as the s o l e c r i t e r i o n f o r i d e n t i f i c a t i o n . Of the o f f s p r i n g o b t a i n e d f rom b a c k c r o s s e s to t r a c h u r u s o n l y 3 7% can be r e c o g n i z e d as such because 32% have counts l i k e those o f t r a c h u r u s . and the r e m a i n i n g progeny (31%) have counts t h a t o v e r l a p w i t h F^ h y b r i d s , B a c k c r o s s i n g , t h e n , a c c o u n t s f o r the t o t a l spect rum of v a r i a t i o n i n p l a t e counts between the r a c e s . G i l l Raker C o u n t s . R e s u l t s o f r a k e r counts ( F i g u r e 9 ) a g a i n demonstrate g e n e t i c c o n t r o l o f the d i f f e r e n c e i n numbers t h a t d i s t i n g u i s h e s the r a c e s . The v a r i a t i o n and mode f o r counts o f o f f s p r i n g o f l e i u r u s and t r a c h u r u s are l i k e those f o r w i l d p o p u l a t i o n s o f each r a c e . Fj_ h y b r i d s are i n t e r m e d i a t e between the r a c e s and are l e s s v a r i a b l e t h a n w i l d h y b r i d s , w h i c h , l i k e p l a t e c o u n t s , c o n f i r m s the presence o f b a c k c r o s s e s i n the h y b r i d z o n e . With b a c k c r o s s e s v a r i a t i o n i s i n c r e a s e d , and the modes f o r r a k e r c o u n t s a re i n t e r m e d i a t e between F-^'s and the r a c e s . A l t h o u g h the range o f v a r i a t i o n i n l a b o r a t o r y r e a r e d h y b r i d s i s l e s s than i n w i l d h y b r i d s r e p e a t e d b a c k c r o s s i n g c o u l d produce t h a t f o u n d i n the w i l d . Counts o f r a k e r s and p l a t e s o f the progeny r e i n f o r c e the c o n c l u s i o n s drawn from the h y b r i d i n d e x , t h a t i n the h y b r i d zone F ' s a re r e l a t i v e l y few and b a c k c r o s s e s to t r a c h u r u s are r a r e . I f they were p r e s e n t t h e y c o u l d be r e c o g n i z e d e i t h e r by F i g u r e 9 , G i l l r a k e r counts o f l a b o r a t o r y r e a r e d ( b l a c k h i s t o g r a m s ) and o f w i l d c a u g h t (hatched his tograms) f i s h p r e s e n t e d f o r c o m p a r i s o n s . See c a p t i o n f o r F i g u r e 8 f o r o t h e r d e t a i l s . CO CO 34 p l a t e or r a k e r c o u n t s . E q u a l l y , the d a t a r e i n f o r c e the c o n c l u s i o n t h a t most o f the b a c k c r o s s e s are to l e i u r u s . Body Depth i n t o S t a n d a r d L e n g t h . The d i f f e r e n c e i n body shape between l e i u r u s and t r a c h u r u s i s under g e n e t i c c o n t r o l ( F i g u r e 10) , b u t i n c o n t r a s t to w i l d f i s h t h e r e i s a n o t i c e a b l e gap between the o f f s p r i n g o f each r a c e . T h i s d i s c r e p a n c y might be c a u s e d by n o n - i s o m e t r i c growth so t h a t a d u l t s o f the r a c e s coverge i n t h i s f e a t u r e ; o r the g r e a t e r v a r i a t i o n i n w i l d a d u l t s may be imposed by e n v i r o n m e n t a l v a r i a t i o n . D o r s a l and A n a l Rays . F i n r a y c o u n t s f o r r a c e s and h y b r i d s are v i r t u a l l y a l i k e . L e i u r u s o f f s p r i n g (sample o f 160) have d o r s a l r a y counts r a n g i n g f rom 10-12 w i t h a mean o f 11.2 (s = 0 . 2 8 3 ) ; f o r t r a c h u r u s o f f s p r i n g (sample o f 80) the range i s 10-13 w i t h a mean o f 11.4 (s = 0 . 8 0 3 ) . F o r a n a l r a y s l e i u r u s have a range o f 7-9 and a mean o f 7.35 (s = 0 . 0 4 8 ) ; t r a c h u r u s have a range o f 7-9 and a mean o f 7.6 (s = 0 . 4 3 5 ) . Hence, the d i f f e r e n c e e n c o u n t e r e d i n the w i l d between r a c e s must be due to e n v i r o n m e n t a l v a r i a t i o n ( p h e n o t y p i c p l a s t i c i t y ) , not g e n e t i c d i v e r g e n c e . I t i s f o r t h i s r e a s o n t h a t d o r s a l and a n a l r a y s were e x c l u d e d from the h y b r i d i n d e x . The r e s u l t s make i t p l a i n t h a t d i f f e r e n c e s between r a c e s (or s p e c i e s ) t o g e t h e r w i t h i n t e r m e d i a t e s cannot be a c c e p t e d w i t h o u t q u e s t i o n as e v i d e n c e f o r h y b r i d i z a t i o n . Other d i f f e r e n c e s o b s e r v e d between r a c e s , though they were not q u a n t i f i e d , i n c l u d e c o l o u r and shape o f s p i n e s . L a b o r a t o r y r e a r e d l e i u r u s r e t a i n t h e i r g r e y i s h - o l i v e c o l o u r m o t t l e d w i t h bands o f brown o r b l a c k , and t h e i r s h o r t s p i n e s , LEIURUS TRACHURUS N=26 N=26 3.1 24 3.7 4 4.3 4.6 4.9 5.2 5.5 5.%// 6.7 7 BODY DEPTH INTO BODY LENGTH LO Figure 10 „ Body depth into standard length of laboratory reared f i s h . See caption, 0 1 Figure 8, for other d e t a i l s . 36 while trachurus remain a bright s i l v e r colour and have long dorsal and pelvic spines. Genetic control of these characters i s thereby indicated. VARIATION WITHIN LEIURUS Plate counts of leiurus i n the L i t t l e Campbell River demonstrate the presence of a c l i n e (Figure 11). The d i r e c t i o n of the c l i n e i s such that mean counts increase with distance upstream so that highest counts are i n the headwaters and lowest counts are nearest the hybrid zone at Station F. The d i r e c t i o n of the c l i n e i s , then, the reverse to that expected i f introgression from the hybrid zone were e f f e c t i n g the character. In addition, linkage relationships between plates and rakers i n wild hybrids was tested with a 2 x 2 contingency table. The number of leiurus with low raker counts and high plate counts and with high raker counts and low plate counts are respectively; 13 to 1 and 2 6 to 6. Only individuals with extreme plate and raker counts are included. With an adjusted chi-square value of 18.98 and one Degree of Freedom P <C ,001 revealing that individuals with low raker counts tend to have high plate counts. 3 4 5 6 7 3 4 5 6 7 3 4 5 6 7 STATION A STA. B STA. C F i g u r e 11 . C l i n a l v a r i a t i o n i n numbers o f p l a t e s o f l e i u r u s . Data are b a s e d on c o u n t s of more than 2000 f i s h . Samples of a t l e a s t 200, and u s u a l l y 400-500 f i s h , were made a t each S t a t i o n . co 38 SEASONAL ISOLATION MATERIALS AND METHODS B r e e d i n g d a t a were g a t h e r e d o v e r the two y e a r s o f t h i s s t u d y to determine what c o n t r i b u t i o n s e a s o n a l i s o l a t i o n made to r e p r o d u c t i v e i s o l a t i o n . The s t u d y was l a r g e l y c o n f i n e d to the L i t t l e Campbel l R i v e r and a d j a c e n t ponds, b u t o b s e r v a -t i o n s were made i n streams on Vancouver I s l a n d i n the summer o f 1965. Throughout the b r e e d i n g season p o p u l a t i o n s were c e n s u s e d a t l e a s t twice w e e k l y . T r a c h u r u s was sampled a t S t a t i o n N and l e i u r u s a t S t a t i o n D and inasmuch as the h e a v i l y w e i g h t e d l e a d l i n e d e s t r o y e d a q u a t i c v e g e t a t i o n and presumably s t i c k l e b a c k n e s t s , s i t e s f o r s e i n i n g were r o t a t e d w i t h i n a one m i l e s e c t i o n o f s t ream a t each S t a t i o n . Numbers o f b r e e d i n g f i s h were e s t i m a t e d from s t a n d a r d 15 f o o t s e i n e h a u l s . U s u a l l y s e v e r a l h a u l s were made and the average c a t c h f o r the s e r i e s was c a l c u l a t e d . R e c o g n i z i n g b r e e d i n g f i s h was s i m p l e because b r e e d i n g males are b r i g h t l y c o l o u r e d and g r a v i d females have enormously s w o l l e n abdomens. The f i n e mesh o f the s e i n e c a p t u r e d r e c e n t l y h a t c h e d f r y so t h a t t h e i r e a r l i e s t h a t c h i n g c o u l d be r e c o r d e d . I t i s assumed t h a t n u p t i a l l y c o l o u r e d males and g r a v i d females are b r e e d i n g f i s h though t h i s may not always be a r e l i a b l e measure . S i n c e g r a v i d females were f o u n d l a t e r i n the s p r i n g than n u p t i a l l y c o l o u r e d males and became n o n - r e p r o d u c t i v e e a r l i e r i n the summer i t seemed most r e a s o n a b l e to r e c o r d the b e g i n n i n g and end o f the r e p r o d u c t i v e 39 season by t h e i r presence and a b s e n c e . The f o l l o w i n g c r i t e r i a were, t h e n , u s e d to i n d i c a t e d i f f e r e n t phases o f the b r e e d i n g s e a s o n : 1. B e g i n n i n g o f r e p r o d u c t i o n : e s t i m a t e d from the f i r s t g r a v i d females c o l l e c t e d i n the s e a s o n . 2. O c c u r r e n c e o f r e p r o d u c t i o n s e s t i m a t e d from the f i r s t f r y c o l l e c t e d i n the s e a s o n . 3. Peak of b r e e d i n g : i n d i c a t e d by t h a t p e r i o d when the l a r g e s t number o f n u p t i a l l y c o l o u r e d males and g r a v i d females were c a p t u r e d . 4. End o f b r e e d i n g s e a s o n : i n d i c a t e d by the l a s t g r a v i d females c o l l e c t e d i n the s e a s o n . RESULTS The L i t t l e Campbel l R i v e r  L e i u r u s and t r a c h u r u s b o t h have b r e e d i n g seasons t h a t last , f o r about f o u r months, b u t l e i u r u s b e g i n s to b r e e d much e a r l i e r i n the y e a r , s t a r t i n g i n l a t e F e b r u a r y o r e a r l y March ( F i g u r e 12). The races are b r e e d i n g a t the same time f o r o n l y one month. However, the o p p o r t u n i t y f o r i n t e r b r e e d i n g i s f u r t h e r r e s t r i c t e d f o r when maximum numbers o f one r a c e are b r e e d i n g minimum numbers o f the o t h e r are b r e e d i n g . D u r i n g the peak o f b r e e d i n g season f o r l e i u r u s t h e r e are v e r y low numbers (<0.2 per s t a n d a r d s e i n e h a u l ) o f e a r l y b r e e d i n g t r a c h u r u s p r e s e n t i n the s t r e a m . C o n v e r s e l y , d u r i n g the peak o f b r e e d i n g season o f t r a c h u r u s , numbers o f b r e e d i n g l e i u r u s have s h a r p l y d e c l i n e d , 40 • I i I I I I MARCH APRIL MAY JUNE JULY AUG. SEPT. F i g u r e 12. B r e e d i n g seasons f o r the r a c e s o f s t i c k l e b a c k i n the L i t t l e Campbel l R i v e r , 1964. Data f o r 1965 § f i r s t b r e e d i n g l e i u r u s , March 2 and f o r t r a c h u r u s May 12; f i r s t f r y o f l e i u r u s , A p r i l 28 and f o r t r a c h u r u s J u l y 7; peak o f b r e e d i n g f o r l e i u r u s , A p r i l 12-26 and f o r t r a c h u r u s June 6 - A u g u s t 2; end o f season f o r l e i u r u s , J u l y 10 and f o r t r a c h u r u s September 18. 41 Data f o r the b r e e d i n g season o f l e i u r u s i n ponds (D and M) resemble t h a t from the s t ream b u t f o r one d i f f e r e n c e . In b o t h ponds l e i u r u s c o n t i n u e s to b r e e d l a t e r i n t o the summer ( i n 1964 u n t i l A u g u s t 5; i n 1965 u n t i l J u l y 16). In p a s s i n g , i t may be ment ioned t h a t p o s t - r e p r o d u c t i v e m o r t a l i t y was v e r y n o t i c e a b l e i n l e i u r u s , w h i c h began i n l a t e June and c o n t i n u e d t h r o u g h o u t J u l y . T h e i r c o r p s e s were e s p e c i a l l y c o n s p i c u o u s i n the p o n d s . Many a d u l t s t h a t were s c a r c e l y a l i v e had e i t h e r f u n g u s e d b o d i e s o r e r u p t i o n s on the c a u d a l p e d u n c l e . Two f a c t o r s may c o n t r i b u t e to t h i s m o r t a l i t y , In ponds temperatures o f 20-27°C were not uncommon d u r i n g l a t e June and J u l y . In the l a b o r a t o r y I have o b s e r v e d t h a t temperatures as h i g h as 24°C are l e t h a l to a d u l t s , A p r e d a t o r , L e t h o c e r u s , was e x c e e d i n g l y abundant i n ponds d u r i n g June and J u l y , and on s e v e r a l o c c a s i o n s I have f o u n d t h i s i n s e c t f e e d i n g on a d u l t s t i c k l e b a c k s . They were f o u n d near the s u r f a c e o f the water i n a q u a t i c v e g e t a t i o n h a n g i n g head-down. Dead s t i c k l e b a c k s were r e p e a t e d l y r e f u s e d , b u t l i v e ones p r e s e n t e d by the t a i l were r e a d i l y t a k e n . H y b r i d s Numerous n u p t i a l l y c o l o u r e d male and g r a v i d female h y b r i d s were c o l l e c t e d i n the h y b r i d zone a n d i n Pond M„ A most u n u s u a l r e l a t i o n s h i p was f o u n d (Table HI), The r e s u l t s o f b o t h y e a r s demonstrate an o b v i o u s s e a s o n a l i s o l a t i o n between the sexes o f h y b r i d s . B r e e d i n g males were t a k e n i n A p r i l and May and g r a v i d females i n June and J u l y . In e f f e c t , t h e n , the 42 T a b l e I I I . Numbers o f b r e e d i n g h y b r i d s t a k e n on dates i n d i c a t e d . BREEDING HYBRIDS, 1964 BREEDING HYBRIDS, 1965 Date o* Date cyr A p r i l 6 9 0 A p r i l 2 17 0 A p r i l 10 23 0 A p r i l 8 21 0 A p r i l 12 13 0 A p r i l 14 20 0 A p r i l 18 18 0 A p r i l 20 9 0 A p r i l 25 41 0 A p r i l 24 8 0 May 3 14 0 A p r i l 29 23 0 May 5 21 0 May 8 15 0 May 13 27 0 May 16 16 0 May 2 7 11 0 May 30 6 7 June 3 0 0 June 3 5 12 June 7 0 14 June 12 0 24 June 9 0 26 June 14 0 27 June 15 0 28 June 24 0 16 June 18 0 18 June 28 0 19 June 2 6 0 15 June 29 0 22 J u l y 5 0 10 43 b r e e d i n g season o f h y b r i d males o v e r l a p s the b r e e d i n g season o f l e i u r u s , and the b r e e d i n g season o f h y b r i d females o v e r l a p s t h a t o f t r a c h u r u s . Pond M i s s m a l l , about t h r e e - f o u r t h s an a c r e , w i t h e x t e n s i v e s h a l l o w s and dense emergent v e g e t a t i o n and s t i c k l e b a c k s were always c o l l e c t e d by s e i n i n g c o m p l e t e l y a round the s h o r e . C o n s e q u e n t l y , i t i s u n l i k e l y t h a t the r e s u l t s are s p u r i o u s . S e i n i n g i n the s t ream i s a l s o e f f i c i e n t , and a g a i n i t seems u n l i k e l y t h a t r e s u l t s are s p u r i o u s , v T h i s p h y s i o l o g i c a l t r a i t seems to be i n h e r i t e d as a s e x - l i m i t e d c h a r a c t e r such t h a t b r e e d i n g season o f l e i u r u s i s dominant i n h y b r i d males and b r e e d i n g season o f t r a c h u r u s i s dominant i n h y b r i d f e m a l e s , S e l a n d e r (1964) has a l s o r e p o r t e d s e x - l i m i t e d c h a r a c t e r s i n h y b r i d b i r d s . E a r l y Spawning T r a c h u r u s A s e p a r a t e s u b s e c t i o n i s d e v o t e d to these i n d i v i d u a l s f o r t h e y make the c h i e f c o n t r i b u t i o n to h y b r i d i z a t i o n i n the L i t t l e Campbel l R i v e r , E a r l y b r e e d i n g i n d i v i d u a l s o f t r a c h u r u s were c o l l e c t e d i n the s t ream b o t h y e a r s . They p r e c e d e d the p r i n c i p a l b r e e d i n g c o n g r e g a t i o n s i n t o the s t ream by as much as one month ( F i g u r e 12 ) . They were f u r t h e r c h a r a c t e r i z e d b y m i g r a t i n g f a r t h e r upstream than i n d i v i d u a l s i n the p r i n c i p a l spawning m i g r a t i o n , b y p a s s i n g the t r a c h u r u s b r e e d i n g grounds ( S t a t i o n N) and the u n i n h a b i t e d c l a y b e l t , thus e s t a b l i s h i n g themselves i n the h y b r i d zone ( F i g u r e 3 ) . So i t i s t h a t these i n d i v i d u a l s were c o l l e c t e d a t S t a t i o n s L , M, and M', i n e a r l y 44 May. A t the end o f May when the p r i n c i p a l spawning m i g r a t i o n s a r r i v e d i n the s t ream they s e t t l e d a t S t a t i o n N, and t h e r e was no n o t i c e a b l e i n c r e a s e o f t r a c h u r u s i n the h y b r i d zone a t t h a t t ime or l a t e r . In May o f 1965 I a t t e m p t e d to c o n f i r m these o b s e r v a t i o n s i n a more c r i t i c a l way. D u r i n g the f i r s t week i n May 1965, a h a l f - i n c h m o n o f i l a m e n t g i l l n e t was suspended a c r o s s the s t ream w i t h i n the c l a y b e l t , between the t r a c h u r u s b r e e d i n g grounds and the h y b r i d zone , and l e f t . Each day the net was s e a r c h e d f o r f i s h and a t o t a l o f n i n e t r a c h u r u s were c a p t u r e d o v e r s i x d a y s . The p r o c e d u r e was r e p e a t e d the second week i n June a f t e r the p r i n c i p a l b r e e d i n g m i g r a t i o n o f t r a c h u r u s h a d a r r i v e d a t S t a t i o n N . None was c a p t u r e d . A g a i n i n 1965 e a r l y spawning m i g r a n t s were c o l l e c t e d i n the h y b r i d z o n e . The f o l l o w i n g d a t a f o r c a t c h e s o f e a r l y and p r i n c i p a l spawnaing m i g r a t i o n s were g a t h e r e d . In 1964, 26 t r a c h u r u s ( e a r l y m i g r a n t s ) were c a p t u r e d d u r i n g the f i r s t t h r e e weeks of May w i t h i n the h y b r i d zone? f o u r i n d i v i d u a l s were t a k e n a t S t a t i o n N . D u r i n g the f i r s t t h r e e weeks of June, 19 t r a c h u r u s were c a p t u r e d i n the h y b r i d zone and thousands were present, a t S t a t i o n N (see T a b l e I I ) „ In 1965, 13 e a r l y m i g r a n t t r a c h u r u s were c a p t u r e d d u r i n g the f i r s t t h r e e weeks o f May w i t h i n the h y b r i d zone? t h r e e were t a k e n a t S t a t i o n N D D u r i n g the f i r s t t h r e e weeks o f June 14 t r a c h u r u s were c a p t u r e d i n the h y b r i d zone and hundreds were p r e s e n t a t S t a t i o n N ( r e c a l l t h a t a d r a s t i c r e d u c t i o n i n d e n s i t y o f t r a c h u r u s o c c u r r e d i n 1965) , A c o m p a r i s o n was made between p l a t e counts o f 45 i n d i v i d u a l s f rom the p r i n c i p a l b r e e d i n g m i g r a t i o n (sample o f 404) and i n d i v i d u a l s from the e a r l y b r e e d i n g m i g r a t i o n (sample o f 3 4 ) . The r a n g e s , means, and v a r i a n c e s are the f o l l o w i n g ; f o r the p r i n c i p a l b r e e d i n g m i g r a t i o n , R = 30-35, X = 32 .7 , s 2 = 0 .993 ; f o r the e a r l y b r e e d i n g m i g r a t i o n , R = 33-36, X = 34 .55 , s 2 = 1 . 7 6 . V a r i a n c e s c o u l d not be assumed e q u a l so t h a t t h e a p p r o x i m a t i o n o f t - t e s t was u s e d ( B a i l e y , 1959) . A v a l u e o f P = .001 was o b t a i n e d , v e r i f y i n g the o b s e r v a t i o n t h a t p l a t e c o u n t s o f e a r l y b r e e d e r s average h i g h e r counts than those from the p r i n c i p a l m i g r a t i o n . To r e c a p i t u l a t e , e a r l y b r e e d i n g m i g r a n t s e n t e r the s t ream about one month b e f o r e the p r i n c i p a l t r a c h u r u s spawning m i g r a t i o n , move f a r t h e r upstream and s e t t l e i n the h y b r i d zone a t s i t e s where h y b r i d swarms have d e v e l o p e d ( S t a t i o n s M and M ' ) , and have s i g n i f i c a n t l y h i g h e r p l a t e c o u n t s . A l l these f a c t s p o i n t to the c o n c l u s i o n t h a t these i n d i v i d u a l s a re g e n e t i c a l l y d i f f e r e n t from i n d i v i d u a l s w i t h i n the p r i n c i p a l b r e e d i n g m i g r a t i o n . Vancouver I s l a n d P o p u l a t i o n s B e g i n n i n g June 15, 1965, an e i g h t day f i e l d t r i p was made to v a r i o u s streams on Vancouver I s l a n d to f i n d o u t i f s e a s o n a l i s o l a t i o n was more w i d e s p r e a d . I f so , i t was p r o b a b l e t h a t l e i u r u s had c o m p l e t e d the b r e e d i n g season and t r a c h u r u s was a t t h e peak o f b r e e d i n g s e a s o n . The r e s u l t s are summarized i n T a b l e IV. The f o u r streams l i s t e d were sampled a t f r e q u e n t i n t e r v a l s f rom the e s t u a r y to the headwaters , b u t no l e i u r u s T a b l e I V . P r o p o r t i o n o f each race b r e e d i n g i n Vancouver I s l a n d s t r e a m s , 1965. Stream and Date L e i u r u s T r a c h u r u s B i g Qual icum R i v e r June 15 Sample s i z e - hundreds B r e e d i n g f i s h - 0 ( f r y to 2 .1 cm abundant i n a d j a c e n t marshes) Sample s i z e - 30 B r e e d i n g f i s h - 26(87%) F r e n c h Creek June 17 None p r e s e n t Sample s i z e - 163 B r e e d i n g f i s h - 126(773 B o n s a l l Creek June 18 Sample s i z e - 96 B r e e d i n g f i s h - 3(3%) Sample s i z e - 72 B r e e d i n g f i s h - 72 Sooke R i v e r June 20 None p r e s e n t Sample s i z e - 86 B r e e d i n g f i s h - 86 47 c o u l d be f o u n d i n two o f the s t r e a m s . A l l p o p u l a t i o n s o f t r a c h u r u s sampled were b r e e d i n g , and as e x p e c t e d no b r e e d i n g p o p u l a t i o n s o f l e i u r u s were f o u n d , A l a n d l o c k e d p o p u l a t i o n o f t r a c h u r u s e x i s t e d i n a s m a l l b e a v e r marsh a d j a c e n t to F r e n c h Creek , and i t s banks were c a r e f u l l y i n v e s t i g a t e d to i n s u r e t h a t the p o p u l a t i o n was c o n f i n e d . About one h u n d r e d t r a c h u r u s were s e i n e d and i n s p e c t e d ? a l m o s t a l l were i n b r e e d i n g c o n d i t i o n . T h i s p r o v i d e s a d d i t i o n a l e v i d e n c e (a long w i t h e v i d e n c e from h y b r i d s ) t h a t the d i f f e r e n c e i n b r e e d i n g between l e i u r u s and t r a c h u r u s i s under s t r o n g g e n e t i c c o n t r o l . F o r i f the d i f f e r e n c e were t r i g g e r e d by e n v i r o n m e n t a l f a c t o r s the t r a c h u r u s t r a p p e d i n f r e s h w a t e r s h o u l d not have been i n b r e e d i n g c o n d i t i o n , as l e i u r u s i n l o c a l f r e s h w a t e r ponds were n o t . In the B i g Q u a l i c u m R i v e r t r a c h u r u s were b r e e d i n g , near the e s t u a r y , and i t i s noteworthy t h a t n i n e i n d i v i d u a l s were t a k e n 7 m i l e s i n l a n d j u s t below H o m e L a k e , These i n d i v i d u a l s , l i k e e a r l y b r e e d e r s i n the L i t t l e Campbel l R i v e r , had u n u s u a l l y h i g h p l a t e counts w i t h a range from 33-36 and a mean o f 35,2, 48 ETHOLOGICAL ISOLATION S p e c i f i c c o u r t s h i p d i s p l a y s t h a t p r o v i d e f o r the c h o i c e of c o n s p e c i f i c mates, and thus p r e v e n t h y b r i d i z a t i o n , c o n s t i t u t e an i m p o r t a n t c a t e g o r y o f i s o l a t i o n mechanisms i n a n i m a l s . When i s o l a t i n g mechanisms are a t i s s u e i t i s e s s e n t i a l t h a t mate p r e f e r e n c e be i n v e s t i g a t e d . R e p r o d u c t i v e b e h a v i o u r o f the t h r e e - s p i n e d s t i c k l e b a c k i s w e l l known, b u t n o t h i n g i s known r e g a r d i n g i t s c o u r t s h i p b e h a v i o u r as an i s o l a t i n g mechanism. MATERIALS AND METHODS Methods d e s c r i b e d by van I r s e l (1953) were e f f i c i e n t f o r h o l d i n g s t i c k l e b a c k s p r i o r to mate p r e f e r e n c e t e s t s . Large numbers o f males o f b o t h r a c e s were h e l d i n a 200 g a l l o n cement tank, each race b e i n g s e p a r a t e d w i t h a wooden p a r t i t i o n t h a t d i v i d e d the tank i n h a l f . A c o n s t a n t f l o w of d e c h l o r i n a t e d t a p - w a t e r p r o v i d e d c i r c u l a t i o n and a n e a r l y u n i f o r m temperature o f 8 ° C w h i l e a c o n t r o l l e d p h o t o p e r i o d gave 16 h o u r s o f l i g h t . The low water temperature and crowded c o n d i t i o n s k e p t f i s h a t a d e c r e a s e d l e v e l o f s e x u a l e x c i t a t i o n and m i n i m i z e d f i g h t i n g . G r a v i d females that, were ready to mate were a l m o s t i m p o s s i b l e to h o l d i n the l a b o r a t o r y f o r more than a day because they would s p o n t a n e o u s l y drop t h e i r eggs , hence , f r e s h females were c o l l e c t e d a t the L i t t l e C a m p b e l l R i v e r j u s t p r e c e d i n g mate p r e f e r e n c e e x p e r i m e n t s . S i x t e e n a q u a r i a (47 cm x 29 cm), each h o l d i n g 8 g a l l o n s , 49 were u n i f o r m l y p r e p a r e d f o r mate p r e f e r e n c e t e s t s . The bot tom was c o v e r e d w i t h a l a y e r of sand 5 cm deep, and a dense c o v e r i n o f R i c c i a a l g a e f l o a t e d on the s u r f a c e . Water temperature i n the a q u a r i a f l u c t u a t e d between 1 7 ° and 1 9 ° C . S i x t e e n o f the most b r i g h t l y c o l o u r e d males were then s e l e c t e d , e i g h t each o f l e i u r u s and t r a c h u r u s , and t r a n s f e r r e d from the h o l d i n g tank to the a q u a r i a . The sudden i n c r e a s e i n temperature and the s h i f t f rom crowded to s o l i t a r y c o n d i t i o n s u s u a l l y p r o d u c e d a r a p i d o n s e t o f r e p r o d u c t i v e a c t i v i t i e s . F i r s t , n e s t s o f a l g a e are c o n s t r u c t e d i n the s a n d . Once a n e s t i s made the male burrows through the b a l l o f a l g a e l e a v i n g an e n t r a n c e and an e x i t and t h i s a c t i o n , c a l l e d " c r e e p i n g t h r o u g h " may be used as an i n d i c a t o r s i g n i f y i n g the t r a n s i t i o n from n e s t b u i l d i n g a c t i v i t i e s to c o u r t s h i p a c t i v i t i e s (van I r s e l , 1953; S e v e n s t e r , 1961) . O n l y a f t e r t h i s a c t i v i t y had been o b s e r v e d were females i n t r o d u c e d f o r t e s t s . In most i n s t a n c e s males c o m p l e t e d n e s t s about t h r e e days a f t e r they were i n t r o d u c e d i n t o the a q u a r i a . Each p a i r of females was c a r e f u l l y s e l e c t e d so t h a t they were as n e a r l y e q u a l i n s i z e and g r a v i d i t y as p o s s i b l e . D i f f e r e n c e i n s i z e between females never v a r i e d more than 3 mm. Once a p a i r o f females was chosen they were p r e s e n t e d to a " t e s t e r " male k e p t s p e c i f i c a l l y f o r t h a t p u r p o s e . I t i s w e l l known t h a t females ready to mate w i l l r e s p o n d to the c o u r t s h i p of a male by p e r f o r m i n g "head u p " ( f a c i n g the male and t i l t i n g h e r body a t a 4 5 ° a n g l e ) . Females to be u s e d i n exper iments were p l a c e d i n g l a s s tubes f i l l e d w i t h water and suspended i n 50 the aquar ium o f the " t e s t e r " m a l e . I f b o t h females responded to the c o u r t i n g male w i t h "head u p " t h i s was t a k e n as a r e l i a b l e i n d i c a t i o n t h a t each female was p r e p a r e d to mate and the e x p e r i m e n t c o n t i n u e d . I f not o t h e r females were t e s t e d u n t i l two r e s p o n s i v e ones were f o u n d . Two f e m a l e s , one each o f l e i u r u s and t r a c h u r u s , t h a t were o f e q u a l s i z e , g r a v i d i t y , and p r e p a r e d n e s s to mate, were then c o n c u r r e n t l y i n t r o d u c e d i n t o the aquarium o f the e x p e r i -mental m a l e . C o u r t s h i p a c t i v i t i e s would b e g i n and e v e n t u a l l y , u s u a l l y w i t h i n 15 m i n u t e s , one o f the females would r e s p o n d to the male , f o l l o w him to h i s n e s t , and d e p o s i t h e r eggs t h e r e . T h i s r e s u l t was r e c o r d e d i n the c l o s e d s e q u e n t i a l t e s t d e s i g n o f C o l e (1962), exper iments c o n t i n u i n g u n t i l s i g n i f i c a n c e or n o n - s i g n i f i c a n c e was a c h i e v e d . A f t e r r e s u l t s were r e c o r d e d f o r a male and the p a i r o f f e m a l e s , the f i s h were d i s c a r d e d and exper iments began i n a n o t h e r a q u a r i u m . Female p r e f e r e n c e t e s t s were a l s o c o n d u c t e d to d e t e c t a p r e f e r e n c e by females f o r males o f the same r a c e . A s e r i e s o f t h r e e 40 g a l l o n a q u a r i a (70 cm x 35 cm) were u s e d as e x p e r i m e n t a l t a n k s . A s t r i p o f sand 5 cm wide was p l a c e d a t b o t h ends o f each aquar ium, and the i n t e r v e n i n g space between the two s t r i p s o f sand was c o v e r e d w i t h c o a r s e g r a v e l . The depth o f sand and g r a v e l was u n i f o r m l y 5 cm. A double g l a s s p a r t i t i o n w i t h a space o f 11 cm between p a r t i t i o n s was p l a c e d e x a c t l y i n the c e n t r e of the aquar ium so t h a t each g l a s s p l a t e was e q u i d i s t a n t f rom the s t r i p s o f s a n d . A u n i f o r m l a y e r of a l g a e c o v e r e d the s u r f a c e o f the w a t e r . 51 Males of approximately equal size (- 3 mm) were introduced into the aquarium, a leiurus on one side of the central p a r t i t i o n s and a trachurus on the other side. The arrangement of substrate forced males to b u i l d nests i n the sand - they never attempted to b u i l d i n gravel - and males were l e f t u n t i l both had b u i l t nests and u n t i l "creeping through" had been observed. A gravid female was selected and introduced between the glass p a r t i t i o n s . Sometimes the female swam rapidl y to the bottom and was not noticed by either male but eventually she would begin to swim about, as females usually did when f i r s t introduced, and males began to court her. The s i t u a t i o n did not arise i n which one male courted while the other male did not court. Once the experiments were under way observations were made by s i t t i n g q u i e t l y 3 m from the aquarium. If after 15 minutes a female had not responded to a male's courtship with "head up" she was replaced with another. When a female responded to either leiurus or a trachurus male with "head up" the r e s u l t was recorded i n Cole's closed sequential test design. Tests were rotated between the three aquaria and continued u n t i l s i g n i f i c a n c e or non-significance was obtained. RESULTS Mate Preference Tests Results of mate preference tests are given i n Table V. The outcome of the f i r s t closed sequential test series for 52 Table V. Results of mate preference tests (above) and female preference tests (below) taken from Cole's closed sequential test design. Mate Preference Tests With leiurus 's Number of times females of each race mated with male. Each set represents results obtained i n one te s t . leiurus i£ = 7 trachurus £ = 6 leiurus °_ = 6 trachurus $ = 6 leiurus ^ = 6 trachurus °_ = 7 leiurus f£ = 7 trachurus = 9 Total leiurus °_ = 26 Total trachurus = 28 With trachurus 's trachurus °_ = 6 leiurus °_ = 6 trachurus °_ = 8 leiurus °_ = 11 trachurus = 9 leiurus °_ = 7 trachurus ^ = 9 leiUrus °_ = 7 T o t a l trachurus .£ = 32 Total leiurus .°. = 31 Female Preference Tests With leiurus °J s Number of times females f i r s t performed "head up" to a male. leiurus 0*^ = 7 trachurus 8 leiurus d 7™ 6 trachurus c f s 7 Total leiurus 0*= 13 Total trachurus o"= 15 n, trachurus & = 8 trachurus o3*^ 11 With trachurus °.'s leiurus 0 *= 10 leiurus 0^= 8 Total trachurus (f- 19 Total leiurus 0^= 18 53 l e i u r u s and t r a c h u r u s males was not s i g n i f i c a n t , i n d i c a t i n g no p r e f e r e n c e b y the mates . However, C o l e (1962) p o i n t s out t h a t i t i s p o s s i b l e to commit a Type I I e r r o r w i t h t h i s t e s t a c c e p t i n g the n u l l h y p o t h e s i s when a s l i g h t p r e f e r e n c e e x i s t s . To m i n i m i z e t h i s e r r o r a s e r i e s o f c l o s e d s e q u e n t i a l t e s t s were r e p e a t e d u n t i l f o u r r e p l i c a t e s h a d been p e r f o r m e d . In each o f t h e s e , as shown, the n u l l h y p o t h e s i s was a c c e p t e d . The outcome from each s e t o f t e s t s was then p o o l e d to p r o v i d e f o r a more p o w e r f u l t e s t , and the r e s u l t s o f these p o o l e d t o t a l s p r e c l u d e the n e c e s s i t y f o r s t a t i s t i c a l t r e a t m e n t f o r p r e s e n t p u r p o s e s . I t may be c o n c l u d e d t h a t females o f e i t h e r race reproduce w i t h males o f b o t h r a c e s r a n d o m l y . F o r i f t h e r e were d i f f e r e n c e s i n c o u r t s h i p b e h a v i o u r between r a c e s t h a t f u n c t i o n e d as i s o l a t i n g mechanisms, t h e n males and females o f the o p p o s i t e r a c e w o u l d , on the average , be s t i m u l a t e d to mate l e s s o f t e n (or not a t a l l ) than males and females o f the same r a c e . Female C h o i c e T e s t s A g a i n i n these e x p e r i m e n t s t e s t s were r e p l i c a t e d because the n u l l h y p o t h e s i s was always a c c e p t e d - i n the f i r s t sequence f o r l e i u r u s and f o r t r a c h u r u s females (Table V ) . P o o l e d t o t a l s f o r l e i u r u s and f o r t r a c h u r u s females o b v i a t e the n e c e s s i t y f o r s t a t i s t i c a l t r e a t m e n t . I t may be c o n c l u d e d t h a t e t h o l o g i c a l i s o l a t i n g mechanisms on b e h a l f o f females do n o t e x i s t , o r i f they do, they e x i s t a t such an e x c e e d i n g l y low l e v e l t h a t they p l a y an i n s i g n i f i c a n t r o l e i n r e p r o d u c t i v e i s o l a t i o n . 54 H y b r i d R e p r o d u c t i v e A c t i v i t i e s No mate p r e f e r e n c e c o u l d be d e t e c t e d by males o r females o f e i t h e r r a c e . I t , t h e r e f o r e , seemed u n l i k e l y t h a t h y b r i d s w o u l d demonstrate a p r e f e r e n c e , and such exper iments d i d not seem worth w h i l e . However, h y b r i d i n v i a b i l i t y i s w e l l known i n many a n i m a l s , and i t was p o s s i b l e t h a t h y b r i d s might not be a b l e to p e r f o r m c o u r t s h i p a c t i v i t i e s p r o p e r l y , b u i l d n e s t s p r o p e r l y , o r p e r f o r m p a r e n t a l c a r e p r o p e r l y . With t h i s i n mind a s e r i e s o f t e s t s were d e s i g n e d u s i n g presumed Fj_ and b a c k c r o s s h y b r i d s . E i g h t males t h a t were presumed F]_'s and f i v e males t h a t were presumed b a c k c r o s s e s to l e i u r u s (see s e c t i o n on M o r p h o l o g i c a l A n a l y s i s ) were c o l l e c t e d and p l a c e d i n a q u a r i a l i k e those d e s c r i b e d f o r mate p r e f e r e n c e t e s t s . In e v e r y i n s t a n c e males s u c c e s s f u l l y b u i l t n e s t s , c o u r t e d , and spawned w i t h female l e i u r u s . F o r a l l these a c t i v i t i e s they seemed normal when compared w i t h male o f e i t h e r r a c e . When a c l u t c h o f eggs h a d been d e p o s i t e d i n a m a l e ' s n e s t h i s a c t i v i t i e s were o b s e r v e d each d a y . H y b r i d s were seen f a n n i n g , n u d g i n g the n e s t , g l u e i n g and o t h e r a c t i v i t i e s . Most o f the eggs had h a t c h e d a t s i x d a y s , and o b s e r v a t i o n s c o n t i n u e d f o r two more d a y s . Males r e t u r n e d f r y t h a t had s t r a y e d f rom the n e s t , a t t a c k e d a probe i n t r o d u c e d i n t o the aquarium, and p u l l e d the n e s t i n t o a l o o s e aggregate o f a l g a e . In a l l these a c t i v i t i e s they seemed n o r m a l . In one case a h y b r i d had two s e p a r a t e c l u t c h e s o f eggs d e p o s i t e d i n h i s n e s t , one d e p o s i t e d on one day, the o t h e r on the f o l l o w i n g d a y . A f t e r the f i r s t c l u t c h had h a t c h e d he 55 was very busy fanning the remaining clutch of eggs and returning wandering fry to the nest. There was no suggestion of hybrid i n v i a b i l i t y or breakdown i n courtship or reproductive a c t i v i t i e s . I had the impression that hybrid males executed parental care more vigorously than did males of either race. This was e s p e c i a l l y noticeable during fanning and returning f r y that had wandered from the nest. F i n a l l y , s i x hybrid females, presumably backcrosses to trachurus, were c o l l e c t e d and t h e i r reproductive a c t i v i t i e s were examined. These females were placed i n aquaria containing trachurus males that had b u i l t nests, one female i n each aquarium. Five of the hybrid females responded to the male's courtship with "head up", and when the male led, the female followed to the nest, entered, and deposited her eggs. One female did not spawn, but no si g n i f i c a n c e i s attached to this r e s u l t because there were frequently cases i n which females of both races f a i l e d to spawn. There were no suggestions of hybrid i n v i a b i l i t y or breakdown i n hybrid females. 5 6 ECOLOGICAL ISOLATION The two races of stickleback occur i n very dissimilar.environments outside the breeding season, leiurus i n freshwater and trachurus i n the sea. Observations at the L i t t l e Campbell River during the breeding season make i t evident that even when the two races are sympatric i n the same stream segregation exists (Figure 2 ) . This suggests that each race i s associated with d i f f e r e n t aspects of the environment producing what seems to be a very powerful i s o l a t i n g mechanism. A series of experiments was therefore designed to te s t for ecological factors that might be important i n maintaining the observed exclusion. DISPERSAL AND TRANSFER EXPERIMENTS MATERIALS AND METHODS Dispersal experiments were c a r r i e d out with large numbers of leiurus by marking, releasing, and recapturing, individuals moved from one part of t h e i r habitat to another. Transfer experiments, on the other hand, were conducted with leiurus and trachurus by transf e r r i n g individuals of each race from t h e i r own habitat to the habitat of the other. For both experiments f i s h were marked by c l i p p i n g the f i r s t dorsal spine at i t s base. This mark proved to be r e l i a b l e and easy to i d e n t i f y . Six weeks before experiments began 20 f i s h had th e i r spines clipped with heavy n a i l c l i p p e r s and were held 57 i n the l a b o r a t o r y , and 15 o t h e r s were c l i p p e d and h e l d i n s c r e e n l i v e - b o x e s i n the s t r e a m . A f t e r f o u r weeks two o f the f i s h h e l d i n the l a b o r a t o r y and none o f those h e l d i n the s t ream d i e d . No s t i c k l e b a c k showed e v i d e n c e o f r e g e n e r a t i n g t h e i r s p i n e s even though those h e l d i n the l a b o r a t o r y had grown. C a p t u r e d s t i c k l e b a c k s e r e c t t h e i r s p i n e s , and the absence o f the f i r s t d o r s a l s p i n e was c o n s p i c u o u s . M o r e o v e r , t h e r e i s no e v i d e n c e t h a t s p i n e s are u s e d i n l o c o m o t i o n and t h e i r absence s h o u l d not h i n d e r m o b i l i t y . D i s p e r s a l exper iments were p e r f o r m e d a t S t a t i o n D, a t y p i c a l l e i u r u s h a b i t a t . There are s e v e r a l m i l e s o f easy a c c e s s w i t h narrow w i d t h and s h a l l o w w a t e r , i d e a l c o n d i t i o n s f o r r e c a p t u r e e x p e r i m e n t s . F o r the same reasons S t a t i o n D and N were u s e d f o r t r a n s p l a n t e x p e r i m e n t s . RESULTS D i s p e r s a l Exper iments S e v e r a l o b s e r v a t i o n s p o i n t i n the same d i r e c t i o n s u g g e s t i n g t h a t l e i u r u s i s a s e d e n t a r y f i s h . F i r s t , a p o p u l a t i o n l o c a t e d a t S t a t i o n E has about 50% of the i n d i v i d u a l s i n f e c t e d w i t h a p a r a s i t e , Neascus , p r o d u c i n g b l a c k s p o t s on the b o d y . A duck farm by the s t ream i s p r o b a b l y r e s p o n s i b l e f o r the heavy i n f e c t i o n s i n c e ducks may a c t as the d e f i n i t i v e h o s t . The h a b i t a t i s i d e a l and l a r g e number o f l e i u r u s are p r e s e n t i n the d e n s e l y v e g e t a t e d s h a l l o w s , y e t w i t h i n 300 meters e i t h e r up o r downstream t h e r e are dense p o p u l a t i o n s o f l e i u r u s 58 and p r a c t i c a l l y none are p a r a s i t i z e d . Second, the c l i n e i n plate numbers occupies the very short distance of 7% miles. This would be expected only i n a very sedentary f i s h . Last, hybrids are confined to an amazingly narrow zone i n the stream. Other factors are no doubt involved, but th e i r l o c a l i z a t i o n to points of hybridization (coexistence of leiurus and trachurus) suggests low dispersal rates. On 19 March 1965, a sample of 2000 leiurus were c o l l e c t e d at Pond L> adjacent to the stream and c a r r i e d immediately to Station D for release. The spines were clipped and f i s h were released into the stream. Seining for recovery of marked f i s h began two days a f t e r t h e i r introduction and was continuous up and downstream from the point of release. The procedure was terminated a f t e r a series of six or more consecutive hauls yielded no marked f i s h . Data (Figure 13) show that v i r t u a l l y no leiurus were recaptured beyond 600 feet from the point of release. Most were recovered within 100 feet up or downstream, and t h i s was so even 30 days l a t e r when the experiment was terminated. Six months l a t e r c o l l e c t i o n s at Station D turned up large numbers of marked l e i u r u s . As leiurus always do, marked f i s h demonstrated a strong tendency to gather i n densely vegetated shallows along the stream, and peaks of density shown i n the frequency -tek®. d i s t r i b u t i o n corresponds with such locations. Transfer Experiments These experiments were undertaken together with others 300- 0 +300 600 300- 0 +300 600 300- 0 +300 600 DISTANCE IN FEET gure 13. Recapture data f o r l e i u r u s t r a n s f e r r e d from Pond D to S t a t i o n D . 60 to seek an explanation to the observed exclusion between races. Experiments began 6 March 1965 ( e a r l i e r than the previous experiments) when 3081 leiurus were c o l l e c t e d at Station D, marked, and releasted at Station N just below the point where Highway 99 crosses the stream. At that time trachurus had not entered the stream. Recapture of marked f i s h began six days after t h e i r release, but not one leiurus was recovered although a distance of one mile up and downstream was seined. Spot seining began at the estuary and continued upstream to Station N but none was found. This unexpected and sudden disappearance was probably a r e s u l t of dispersal from the area because f i s h , l i k e salmon, that could be predators were present as f r y , and no other predators were known i n the area. Another transplant was attempted on 14 March using 6000 leiurus, and recapture began the following day. Very few individuals were recovered even on the f i r s t day af t e r release (Figure 14), and a f t e r three days only three leiurus were taken over a one mile section of stream. The data suggest that most of the f i s h moved upstream despite a current of 92 cm a second they had to forge while passing through a 230 foot conduit beneath Highway 99. The r e s u l t s of these experiments contrast v i v i d l y with dispersal experiments i n which leiurus was moved from one part of i t s habitat to another. Several prominent changes occur at Station N between ear l y spring and early summer. At the time the foregoing experiments were performed the area i s devoid of vegetation, and current i s swift there being no p l a c i d backwaters along DISTANCE IN F E E T F i g u r e 14. Recapture data f o r the t r a n s f e r of l e i u r u s from S t a t i o n D to S t a t i o n N , <r> Zero r e p r e s e n t s p o i n t of r e l e a s e . See t e x t f o r f u t h e r d e t a i l s . 62 the margins of the stream, i n early June dense beds of Elodea cover the bottom and the current i s m i l d 0 / This seasonal difference at Station N c a l l e d for s t i l l another transplant experiment. On 31 May, 3000 leiurus were marked and released. A population was successfully founded and 24 days l a t e r when recapture stopped large numbers were s t i l l present (Figure 15). Recovery for marked f i s h continued at monthly in t e r v a l s and they remained abundant u n t i l 7 December when none was caught. By that time the Elodea had died down and the current was again f a s t . The r e s u l t s demonstrate a very noticeable s h i f t upstream i n the d i s t r i b u t i o n pattern from the point of release, which persisted throughout the summer. As res u l t s of the dispersal experiment had shown, leiurus proved to have a low dispersal, s e t t l i n g within 600 feet from the point of release and g r a v i t a t i n g toward l o c a l i t i e s where vegetation was most dense and current l e a s t . By 2 June trachurus were present at Station N, and the two races existed sympatrically for the rest of the summer (but r e c a l l that density of trachurus was very reduced i n 1965). Extensive c o l l e c t i o n s were not possible but those that were made suggest a marked increase i n the incidence of hybridization as a r e s u l t of the a r t i f i c a l introduction as can be seen i n the following c o l l e c t i o n data: September 18, 17 hybrids; October 12, 21 hybrids; December 7, 6 hybrids. Over a two year period p r i o r to the introduction only 24 hybrids were c o l l e c t e d at Station N. Reciprocal transplants, moving trachurus from Station DISTANCE IN FEET F i g u r e 15. Recapture d a t a f o r l e i u r u s t r a n s p l a n t e d from S t a t i o n D to S t a t i o n N . Zero r e p r e s e n t s p o i n t o f r e l e a s e . 64 N to Station D, began on 7 June but marking the f i s h was not necessary inasmuch as trachurus were unknown i n the upper reaches. Because trachurus were far less abundant i n 1965 only 350 were c o l l e c t e d and transplanted. Recapture began two days l a t e r . The c h a r a c t e r i s t i c upstream s h i f t i n d i s t r i b u t i o n can again be seen (Figure 16) but did not p e r s i s t . Trachurus dispersed over a greater distance than leiurus so that a f t e r nine days they had moved at l e a s t 1000 feet downstream but just a short distance upstream. There was a continual reduction i n the numbers caught and by 2 7 June few remained. A one mile stretch was again seined on 19, 20, 21 July and none was captured, nor was any found while spot seining many s i t e s i n the upper reaches of the stream. The continual reduction i n numbers of trachurus at Station D together with l i t t l e d i spersal upstream and increasing numbers downstream indicates a downstream migration from the area. During the 20 days that trachurus remained at Station D they exhibited a strong preference for the few l o c a l i t i e s where gravel was present and where the current was mild (most of the current i s sluggish during the summer i n the headwaters). Invariably the largest numbers were seined over such places giving r i s e to temporary clus t e r s of trachurus that lodged i n a very d i f f e r e n t portion of the habitat than did l e i u r u s . I t i s u n l i k e l y that competition played a role i n t h i s habitat exclusion such that resident leiurus forced trachurus into those areas, for 14,000 leiurus had been c u l l e d from the area e a r l i e r i n the spring. Active s e l e c t i o n of these s i t e s 100 r 50 JUNE 9 600 400 200- 0 +200 400 800 600 400 200-JUNE 13 +200 400 ^50 & 0 JUNE 16 800 600 400 200- 0 +200 400 JUNE 20 800 600 400 200- 0 +200 400 50 JUNE 27 800 600 400 200- 0 +200 400 DISTANCE IN FEET F i g u r e 61. Recapture data f o r t r a c h u r u s t r a n s p l a n t e d from S t a t i o n N to S t a t i o n D . Zero r e p r e s e n t s p o i n t of r e l e a s e . U l 66 seems more l i k e l y . Many of the trachurus that were caught seemed very unhealthy. Eleven dead f i s h were found and males l o s t t h e i r b r i l l i a n t nuptial colours - which a l l introduced males had -while females assumed a d u l l s i l v e r colour rather than the bright s i l v e r that i s t y p i c a l . They were also emaciated. Mort a l i t y as well as dispersal from the area probably accounted for the continual reduction i n numbers. In summary, dispersal experiments with leiurus confirm observations that t h i s i s a very sedentary f i s h which congregates where vegetation i s dense and current n i l . When i t i s introduced into an environment devoid of vegetation with no r e t r e a t from current i t r a p i d l y disperses from such an inhospitable habitat. Trachurus introduced into a leiurus habitat moves into l o c a l i t i e s of gravel and current eventually disappearing from the area p a r t l y as a r e s u l t of downstream migration and p a r t l y as a r e s u l t of mortality. The inappropriateness of the habitat i s demonstrated by t h e i r generally poor condition. PREFERENCE TESTS A series of preference tests was conduced i n the laboratory i n an attempt to define some of the components of the environment that were important i n securing ecological i s o l a t i o n . Four components seemed p a r t i c u l a r l y important. F i r s t , leiurus were always observed nesting on a mud bottom 67 and t r a c h u r u s on a sand b o t t o m . Second, l e i u r u s n e s t s were always f o u n d among b r o a d - l e a v e d v e g e t a t i o n and t r a c h u r u s n e s t s among E l o d e a or M y r i o p h y l l u m . T h i r d , l e i u r u s d w e l l s i n s t a n d i n g water and n e s t s were f o u n d t h e r e , w h i l e t r a c h u r u s f r e q u e n t s and n e s t s i n g e n t l y f l o w i n g w a t e r . F o u r t h , l e i u r u s i n h a b i t s t e a c o l o u r e d water and t r a c h u r u s c l e a r w a t e r . A l l f o u r components may be i m p o r t a n t inasmuch as they may serve to s e p a r a t e the r a c e s i n t o d i f f e r e n t h a b i t a t s w i t h i n the s t ream thus p r o v i d i n g f o r r e p r o d u c t i v e i s o l a t i o n (and c o m p e t i t i v e e x c l u s i o n ) . MATERIALS AND METHODS S u b s t r a t e P r e f e r e n c e T e s t s In F e b r u a r y s t o c k s o f t r a c h u r u s were c o l l e c t e d f r o m wharves i n the sea .near the e s t u a r y o f the s tream and h e l d i n the l a b o r a t o r y i n 40 g a l l o n a q u a r i a s u p p l i e d w i t h c i r c u l a t i n g sea w a t e r , a temperature o f 8°C, and a p h o t o p e r i o d o f 8 hours o f l i g h t a d a y . L e i u r u s were a l s o t a k e n and h e l d the same e x c e p t i n f r e s h w a t e r . On 1 A p r i l the p h o t o p e r i o d was i n c r e a s e d to 16 h o u r s a day and the temperature was g r a d u a l l y e l e v a t e d to 18°C. A dense l a y e r o f a l g a e was s u p p l i e d to a l l a q u a r i a . These c o n d i t i o n s promote s e x u a l m a t u r i t y (Baggerman, 1957). On 15 A p r i l t r a c h u r u s were t r a n s f e r r e d to f r e s h w a t e r , and b y 28 May many l e i u r u s and t r a c h u r u s had a t t a i n e d n u p t i a l c o l o u r s . Because s t o c k s were h e l d o v e r g l a s s - b o t t o m e d a q u a r i a t h e r e c o u l d n o t be any r e c e n t h a b i t u a t i o n to e i t h e r a sand o r a mud 68 s u b s t r a t e . S i x 8 g a l l o n a q u a r i a were p r e p a r e d f o r s u b s t r a t e p r e f e r e n c e t e s t s , mud f r o m S t a t i o n D a n d s a n d f r o m S t a t i o n N p r o v i d i n g t h e m a t e r i a l s . A q u a r i a were d i v i d e d i n h a l f w i t h a p l a t e o f g l a s s a n d on one s i d e a l a y e r o f mud was p l a c e d , on t h e o t h e r a l a y e r o f s a n d a l l u n i f o r m l y a t a d e p t h o f 5 cm. The a q u a r i a were t h e n f i l l e d w i t h w a t e r an d a f t e r s e d i m e n t h a d s e t t l e d t h e p a r t i t i o n s were removed an d a l a y e r o f a l g a e p l a c e d on t h e s u r f a c e . V e g e t a t i o n P r e f e r e n c e T e s t s The same b a s i c d e s i g n o f a q u a r i a a n d s t o c k s o f f i s h were u s e d i n t h e s e e x p e r i m e n t s . The a q u a r i a were p l a n t e d h a l f w i t h E l o d e a a n d h a l f w i t h P e n a n t h e , a h e a d w a t e r p l a n t i n w h i c h l e i u r u s f r e q u e n t l y n e s t s . A s p a c e o f 10 cm was l e f t open on t h e o b s e r v e r ' s s i d e and v e g e t a t i o n was p l a n t e d i n p a r a l l e l rows 4 cm a p a r t t o f a c i l i t a t e o b s e r v a t i o n . C u r r e n t P r e f e r e n c e T e s t s An 8 g a l l o n a q u a r i u m was d i v i d e d i n h a l f w i t h a p l a t e o f f r o s t e d g l a s s s u c h t h a t one e n d was f i r m l y f i t t e d a g a i n s t one s i d e o f t h e a q u a r i u m a n d t h e o t h e r e n d f e l l s h o r t o f t h e o p p o s i t e s i d e o f t h e a q u a r i u m b y 12 cm. The b o t t o m o f t h e f r o s t e d p l a t e was f i r m l y wedged i n 8 cm o f s a n d . The t o p o f t h e p a r t i t i o n p r o j e c t e d above t h e s u r f a c e o f t h e w a t e r s o t h a t t h e e f f e c t s o f a c u r r e n t g e n e r a t e d on one s i d e o f t h e p a r t i t i o n d i d n o t d i s t u r b w a t e r on t h e o t h e r s i d e . 69 The open end permitted free passage of a f i s h from one side to the other of the aquarium, A variable speed e l e c t r i c a l l y driven motor with a 2,5 cm propeller was suspended just beneath the surface i n a corner of the aquarium at the open end, and the device was held firm with a clap and a r i n g stand. At the b l i n d ends of the aquarium mesh baskets with open bottoms were suspended and f i l l e d with algae. On the bottom at the open end of the aquarium pebbles were planted. This produced a tank i n which f i s h could f r e e l y choose to nest i n either current or standing water. In the open end where a s l i g h t current was detected pebbles prevented sticklebacks from b u i l d i n g nests. Fine threads or pieces of brine shrimp sank d i r e c t l y to the bottom on one side, but on the other side such items flowed gently through the water. Males placed i n the current tank quickly learned to pass f r e e l y from side to side and there was no i n d i c a t i o n that they avoided the propeller, which at any rate was aside and i n a corner. Before and during nesting trachurus spent a great deal of time r i d i n g the current just i n front of the rotor. Water Quality Preference Tests For these experiments tea-coloured water from Station D and c l e a r water from Station N was transported to the laboratory. The apparatus and methods used were l i k e those described by Mclnerney (1964) for s a l i n i t y preference t e s t s . In h i s experiments one chamber was f i l l e d with a given concentration of s a l t water, and freshwater from the adjacent 7 0 chamber was allowed to r i s e above a central p a r t i t i o n forming a bridge of water. The d i f f e r e n t i a l densities of s a l t and freshwater maintained the two separate while the bridge of water provided free passage so that the f i s h could choose between s a l i n i t i e s . In my experiments i t was necessary to determine how much mixing occurred when water of equal density was placed i n both chambers. A series of tests were made by dyeing water i n the plugged chamber with Methyl Blue and then flooding to form a bridge. A very small portion of the dye moved into the flooded chamber as a r e s u l t of turbulence, but the two kinds of water c l e a r l y remained separate. RESULTS Substrate Preference Tests Three n u p t i a l l y coloured male leiurus and trachurus were removed from the stock tanks and introduced, one each, into the six prepared aquaria. They were l e f t u n t i l they had b u i l t nests and were then replaced by other males. Each time a substrate was selected by males of either race the choice was entered i n Cole's closed sequential test design and tests continued u n t i l significance or non-significance was achieved. When sticklebacks b u i l d nests they dig a p i t i n the sand and l a t e r carry algae to the p i t glueing t h i s together with a secretion, presumably from the kidney. It may be that 71 secretions around the nest influence s i t e s chosen by subsequent males so the substrate around and beneath the nest was always removed a f t e r each experiment. Results are highly s i g n i f i c a n t , leiurus demonstrating a strong preference for nesting on mud (7:0) and trachurus a strong preference for nesting on sand ( 9 s l ) . Vegetation Preference Tests  Leiurus males show a preference for nesting among Qenanthe (17-4) and trachurus males a preference for nesting among Elodea (9-rl) . Males twice b u i l t nests between rows of Elodea and Qenanthe and the results were discarded. The structure of the vegetation and i t s arrangement i n the aquarium complicates drawing conclusions from the experiment. Qenanthe has long stalks free of leaves with a dense canopy of leaves above, but Elodea has small leaves that clu s t e r densely along the entire length of the st a l k . Thus, among Qenanthe the bottom i s r e l a t i v e l y open and there i s a dense canopy 10 to 15 cm above the bottom. Among Elodea the cover above i s less but the bottom (where the nests are found) of the tank i s much more secluded. Hence, rather than demonstrating a preference for type of vegetation i t i s possible that the two forms are demonstrating a preference for either a more open or a more secluded nesting s i t e . Observations i n the f i e l d , to be described, insinuate the l a t t e r p o s s i b i l i t y . 72 Current Preference Tests Each race demonstrates a strong preference for nesting i n standing water (7:0 for each), but t h i s conclusion does not preclude any importance of current i n segregating the races i n t h e i r choice of a nesting l o c a t i o n . Leiurus very r a r e l y moved into the hal f of the aquarium with current and then only for a b r i e f time. Before nesting began trachurus was most often i n the h a l f with current, and even a f t e r i t had b u i l t i t s nests i n the h a l f with standing water i t s a c t i v i t i e s aside from nest b u i l d i n g were la r g e l y spent i n moving water. These observations w i l l be considered further i n a l a t e r sub-section. Water Quality Preference Tests Equal numbers of f i s h were placed i n each chamber, one containing tea-coloured water and the other containing cl e a r water from Station N. A f t e r flooding, the numbers i n each chamber were recorded at 15 minute i n t e r v a l s . S t i c k l e -backs do not school during the breeding season so that s o c i a l aggregation probably did not bias the r e s u l t s . Preliminary experiments with stocks of f i s h that had been kept i n the laboratory i n c l e a r water revealed a strong preference by both races for clear water. Fresh stocks of leiurus (Station D) and trachurus (Station N) were c o l l e c t e d and used i n l a t e r experiments (Table VI). Again each race, i n both r e p l i c a t e s , exhibits a strong preference for clear water (Station N) which became evident at 45 minutes and persisted for the duration of the experiment. 73 Table VI. Preference tests with stream water. Number of f i s h i n each compartment (tea coloured, clear) at indicated times following introduction to apparatus. Leiurus #1. 30 f i s h tea coloured water Station D clear water Station N #2. 20 f i s h tea coloured water clear water Time - minutes 15 30 45 60 75 90 105 120 11 12 10 3 2 3 19 18 20 27 28 27 29- 28 7 4 1 1 3 7 4 3 13 16 19 19 17 13 16 ,17 Trachurus #1. 30 f i s h tea coloured water clea r water 27 13 9 7 3 4 7 2 3 17 21 23 27 26 23 28 #2. 30 f i s h tea coloured water clear water 16 17 12 6 3 1 0 0 14 13 18 24 27 29 30 30 74 ECOLOGICAL OBSERVATIONS ON VANCOUVER ISLAND Observations on the effectiveness of ec o l o g i c a l i s o l a t i o n were extended to four small streams on Vancouver Island (Table IV). The streams were thoroughly sampled from the headwaters to the estuary with a seine. Results may best be treated i n summary. Estuaries of a l l four streams have b i o t i c and gross physical c h a r a c t e r i s t i c s very much l i k e the estuary of the L i t t l e Campbell River, including a sandy bottom, mixohaline waters (recorded with hydrometer), dense beds of Ulva. and the same species of f i s h . Breeding populations of trachurus were present i n the estuaries of a l l four streams. French Creek, Big Qualicum, and Sooke River are rather s w i f t l y flowing streams with rocky bottoms and l i t t l e vegetation. Sticklebacks were never found where these features p r e v a i l . As mentioned before, nine trachurus were seined i n the headwaters of Big Qualicum, a l l breeding males, and nests were found among beds of Myriophyllum (see next subsection). The habitat had a sandy substrate, a mild current, and some Myriophyllum. Also mentioned e a r l i e r was the beaver pond near the headwaters of French Creek that held a landlocked population of trachurus. The habitat was a t y p i c a l for that race, for the impounded water was choked with broad-leaved vegetation such as Nuphar, Oenanthe. and Potamogeton, the bottom was mud, the water was dark tea colour, and there was no current. A dried stream bed l e d into French Creek, testimony that the waters once fed the creek. At one time trachurus may have migrated each year 75 to spawn i n the s i t e now occupied by the impounded waters, but were trapped there upon completion of the dam. From a sample of 46 individuals three were found that had plate counts l i k e those of hybrids - 15, 16, and 2 7. That they were hybrids i s questionable. The swift waters of French Creek are an unfavourable habitat for leiurus and none was found throughout i t s length, nor were any found i n the entire area. Typically, where leiurus i s present i t i s an abundant f i s h , e s p e c i a l l y so i n ponds. The region i s remote and i t i s not l i k e l y that man had recently introduced leiurus, and i f leiurus had vanished from the pond afte r hybridizing, i t s disappearance must have been recent and complete because sticklebacks have a short l i f e span. Leiurus were p l e n t i f u l only i n habitats with mud bottom, dense aquatic vegetation, standing or very p l a c i d and tea coloured water. Although they were absent from most streams they were abundant i n ponds and marshes. Bonsall Creek i s a small stream about h a l f the length of the L i t t l e Campbell River and the d i s t r i b u t i o n of races there i s worthy of special attention. A large breeding congregation of trachurus was found 1% miles from the estuary where there was a sandy bottom, a mile current, and beds of Elodea and Mvriophyllum. Within 200 feet upstream the habitat changed to mud bottom with dense stands of Potamoqeton pulcher. Qenanthe, and Campanula. In that habitat leiurus alone was c o l l e c t e d . The s h i f t i n habitats along with a complete switch i n races over so b r i e f a distance was compelling evidence supporting ec o l o g i c a l i s o l a t i o n . 76 More f o r c e f u l l evidence yet came from the intervening section where hybrids were common. In thi s zone the edges of the stream were muddy, heavily vegetated, and without current, and leiurus was most common, while i n the centre of the stream there was sand, Elodea and Myriophyllum. and current. Trachurus were captured there i n large numbers. Hybrids were common throughout this section composed of intermingled habitats but not one was found just upstream or downstream although large samples were taken. From preserved samples of 82 f i s h i n the hybrid zone 21% were hybrids; and from a sample of 224 leiurus taken 400 feet upstream and 183 trachurus downstream none were hybrids. i NESTING.SITES AND MATERIALS Data for the loc a t i o n of nesting s i t e s and the materials used to construct nests were gathered at Station D for leiurus and at Station N for trachurus. C l a r i t y of the water and aggressive behaviour of males with nests made them s u r p r i s i n g l y easy to f i n d by s i t t i n g on the banks and watching for males that chased other sticklebacks or f r y of coho salmon. Eventually such males would return to th e i r nests to fan or to carry on with i t s f a b r i c a t i o n . When a nest was found i t was removed and placed i n a v i a l of isopropyl alcohol. Depth of water, substrate, current, temperature, nearby vegetation, and location i n the stream were recorded. Nests were l a t e r placed under a binocular d i s s e c t i n g microscope and the materials used i n the i r construction were recorded. The following results were obtained from 26 leiurus 77 nests: nests were always placed on a, mud bottom and usually situated so that no current could be detected i n the v i c i n i t y ; average temperature was 21°C with a range of 16° to 23°C; nests were usually close to the banks and occasionally within 6 cm of the waterline with an average depth of 24 cm and sometimes as shallow as 4 cm; they were most commonly i n pockets or indentations along the banks near logs or s t i c k s , and although they were in v a r i a b l y found i n open they were always close by stands of Qenanthe, Potamogeton, Nuphar, or Carex. Results from a sample of 25 trachurus nests were the following: always on a sand bottom and so placed that currents ranging from 3 to 6 cm a second passed over the s i t e ; temperature averaged 21°C with a range from 14° to 16°C; nests were usually near the centre of the stream i n water with an average depth of 50 cm; they were always either within or on the downstream side of dense patches of Elodea. Nests of trachurus were s l i g h t l y r a i s ed from the substrate and had more noticeable entrances than those of leiurus because of the contrasting background of l i g h t l y coloured sand. Materials used i n the f a b r i c a t i o n of nests were often decomposed and could be i d e n t i f i e d to a very general category only. I could f i n d no differences whatsoever i n the kinds of material used by each race, which consisted of plant f i b e r s , grass roots (usually fresh), small b i t s of leaves, stems, and twigs. Leiurus nests were always found i n the open where current was absent but trachurus nests were sheltered within or 78 on the downstream side of thickets of Elodea. It i s possible then that the difference found between leiurus and trachurus i n the vegetation preference tests represents a preference for more open or more secluded nesting locations. TROPHIC ADAPTATIONS Stomachs of adults of both races were examined for possible differences i n feeding that might explain the e c o l o g i c a l exclusion between races. A sample of 30 individuals of each race was c o l l e c t e d i n July. Stomachs that were empty or that had food which could not be i d e n t i f i e d were discarded, and others were selected whose contents could be i d e n t i f i e d . Samples were taken on the same days at Station D for leiurus and Station N for trachurus. Contents of the stomach were placed i n a single layer on a g r i d divided into 5 mm squares, and food items were sorted into common l o t s . The percentage of each item was estimated for every stomach and the figures obtained for a l l stomachs were then summed and divided by t o t a l number of stomachs investigated. This method adequately expresses the food habits of a population for a given time and place. Stomach contents were divided into three categories: bottom dwelling organisms, f l o a t i n g or free swimming organisms, and those that may f a l l into e i t h e r of the two previous categories, and therefore, c a l l e d uncertain. Results suggest that leiurus feeds to a large extent on botton dwelling organisms (78%) and that trachurus feeds on f l o a t i n g or swimming organisms 79 (40%). However, trachurus eats a large amount of Chironomidae, which must be c l a s s i f i e d as uncertain because they may be free swimming or attached (Figure 17). The v a l i d i t y of the difference i n feeding habits may be questioned since the two races were sampled from d i f f e r e n t habitats - habitats that may be expected to accommodate d i f f e r e n t invertebrate faunas(Table I ) . Palaemonetes, for example, i s common at Station N during June and July but i s unknown from the upper reaches while Musculium, zygoptera, and coleoptera larvae are common at both Stations yet they are important food items for leiurus only. Even when food items are commonly found i n the two habitats one cannot be ce r t a i n that they are equally available i n both. However, the differences found imply a difference i n feeding behaviour, one taking food l a r g e l y from the bottom, and the other food suspended i n the water. % 0 10 20 30 20 10 0 2 0.1*1 ft: Lu i — CO .^ N 2 UJ o a ! . TRACHURUS LEIURUS UJ 5 U j a . i UJ UJ § U j i o U j Q C: 1 8 Q O C L 1 S o U j Q o U j 5 1 i to o t r . U j c i Q O i BOTTOM DWELLERS UNCERTAIN F i g u r e 17. Food items taken from stomachs o f l e i u r u s and t r a c h u r u s . e x p l a n a t i o n . SUSPENDED See t e x t f o r oo o 81 POSTMATING ISOLATING MECHANISMS Postmating isolating mechanisms include a host of factors that come into play after gametes have been shed and, therefore, do not prevent wastage of gametes as premating mechanisms do. The isolation springs from either genetic or chromosomal incompatibility between populations and may produce any of the following: gamete mortality, zygote mortality, hybrid inferiority, hybrid sterility, or hybrid breakdown. Any of these may serve to either block gene flow between populations or produce hybrid offspring that are at a selective disadvantage as compared to either parental type. MATERIALS AND METHODS Parents for crosses were taken from allopatric populations or from the hybrid zone, and eggs were usually fertilized on the same day that fish were collected. Artifical fertilization was produced by stripping eggs into a small moist petri rfish. A male's testes were immediately removed and placed in a watch glass with a few drops of tap water, chopped into fine pieces with forceps and probe, and poured over the eggs as the container was gently rotate d. Eggs were left to harden for 20 minutes and were then observed through a binocular dissecting microscope to assure that the perivitelline membrane had separated from the egg, as an indication of successful fertilization. Mucous around the egg mass was removed by placing the tip of a dropper into the centre of the mass and pressing 82 i t against the bottom of the dish. Suction from the release of the bulb successfully removed the mucous and adhering b i t s of tissue and sperm. Treated i n t h i s way eggs were much less.prone to attack by fungus. F e r t i l i z e d eggs were transferred to cup-shaped r a f t s of c e l l u l o i d mesh that f l o a t e d on the surface i n jars holding 500 cc of dechlorinated tap water. Each jar was vigorously aerated causing the r a f t of eggs to slowly rotate just beneath the surface. Eggs were incubated at a constant temperature of either 14° or 18°C and under a d a i l y photoperiod of 16 hours. If fungus began to develop on eggs they were treated with a 1:200,000 d i l u t i o n of Malachite Green solution for 6 hours. Once the eggs hatched the contents of the jar were transferred to an enamel tray so that the numbers of f r y and unhatched eggs could be counted. When hybrid crosses were made the egg mass of each female was s p l i t into approximately equal halves. One h a l f of the eggs was f e r t i l i z e d with homoganetic sperm to serve as a control, and the other h a l f was f e r t i l i z e d with heterogametic sperm and each hybrid cross was compared with i t s c o n t r o l . Thus, other causes of egg mortality were not at t r i b u t e d to gametic incompatibility. Hybrid f r y and controls were kept under uniform conditions, but because of l i m i t e d f a c i l i t i e s the d i f f e r e n t combinations of crosses extended over two summers. The classes of o f f s p r i n g were held i n separate gauze baskets 22 cm x 22 cm, and 16 cm deep with about 200 f i s h i n each basket; a l l baskets 83 were suspended i n a 200 gallon cement tank. Dechlorinated tap water c i r c u l a t e d through the tank constantly, holding the temperature between 17° and 19°C. Newly hatched f r y were fed in f u s o r i a as soon as the yolk sac was absorbed and at four days they began to feed on n a u p l i i of l i v e brine shrimp. A f t e r eight weeks they were fed frozen brine shrimp u n t i l the experiments were abandoned at 3% months. RESULTS Survival to Hatching Comparing hatching success of re c i p r o c a l hybrids at both temperatures with t h e i r controls shows that the su r v i v a l of hybrids to hatching approximates the s u r v i v a l of controls. There i s no in d i c a t i o n of genetic incompatibility (Table VII). Backcrosses were made with hybrid males c o l l e c t e d from the hybrid zone. During mid-May backcrosses to leiurus were achieved and at the same time adequate hybrid males were taken and held i n the laboratory to provide for backcrosses to trachurus i n early June. When these backcrosses are compared with t h e i r controls i t may be seen that hatching success of backcrosses i s l i k e that for controls, and there i s no suggestion of genetic incompatibility or hybrid breakdown (Table VII). A r e s t r i c t e d number of backcrosses to leiurus with hybrid females was also made as well as a l i m i t e d number of F2 crosses (seasonal i s o l a t i o n between sexes of hybrids made i t d i f f i c u l t to c o l l e c t both sexes at once). Controls were not Table VII. Hatching success of hybrid crosses. Numbers i n parenthesis represent plate counts of parents. Controls presented i n l e f t hand column and corresponding hybrid crosses i n r i g h t hand column. Leiurus 2 x Leiurus <f Leiurus °- x Trachurus err 1. £(5) x #(4). Hatch =31 Die = 7 Total no. eggs = Mortality = 18% 38 1. %(5) x (7(31) . Hatch =29 Die = 4 Total no. Morta l i t y eggs = = 12% 33 2. °-(3) x 5(5). Hatch = 35 Die = 2 Total no. eggs = Mortality = 5% 37 2. •°-(3) x 5(33). Hatch * 42 Die = 0 Total no. Morta l i t y eggs = = 0% 42 3. $(5) x 5(5). Hatch = 38 Die = 7 Total no. eggs = Mortality = 15% 45 3. £(5) x 5(34). Hatch = 62 Die = 6 Total no. Mortality eggs = = 9% 68 4. ?(4) x 3(5). Hatch = 51 Die = 4 Total no. eggs = Mortality = 7% 55 4. ?(4) x 3(33). Hatch = 45 Die = 2 Total no. Mortality eggs = = 4% 47 5. ?(6) x 5(5). Hatch = 59 Die = 4 Total no. eggs = Mortality = 6% 63 5. ?(6) x 3(32). Hatch =46 Die = 2 Total no. Mor t a l i t y eggs = = 4% 49 6. $(5) x 5(6). Hatch = 47 Die = 2 Total no. eggs = Mortality = 4% 49 6. $(5) x 5(34). Hatch =58 Die = 3 Total no. Mo r t a l i t y eggs = = 5% 61 7. £(4) x 5(5). Hatch =35 Die = 3 Total no. eggs = Mortality = 8% 38 7. £(4) x 0^(32) . Hatch = 46 Die = 2 Total no. Mortality eggs = = 4% 49 TOTAL MORTALITY = 8.9% TOTAL MORTALITY = 5.8% 01 Leiurus x Leiurus 1. $(4) x d*(4). Total no. eggs = 58 Hatch = 57 „ . , 0 o/ Die = 1 Mortality = 2% 2. £(4) x <?(5). Total no. eggs = 52 Hatch = 50 „ . . . . A o, Die = 2 Mortality = 4% 3. ?(5) x 3{5). Total no. eggs = 38 Hatch = 31 -,00/ D £ e _ 7 Mortality = 18% 4. $(5) x <?(4). Total no. eggs = 42 Hatch =41 Die = 1 Mortality = 2% 5. °.(6) x cT(4). Total no. eggs = 47 Hatch = 43 .„ . , ., Die = 4 Mortality = 8% 6. #(4) x 3»(5). Total no. eggs = 31 Hatch = 28 . , . . , rto, D ^ e _ 3 Mortality = 10% 7. $(5) x <?(4). Total no. eggs = 56 Hatch = 54 *i • A. Die = 2 Mortality = 3% TOTAL MORTALITY = 6.1% Leiurus °1 x Trachurus o*^  1. £(4) x 0^(35). Total no. eggs = 34 Hatch = 33 „ . , . . Die = 1 Mort a l i t y = 3% 2. £(4) x 6^33). Total no. eggs = 42 Hatch = 39 „ . . . . Die = 3 Mort a l i t y = 7% 3. $(5) x <?(32). Total no. eggs = 37 Hatch = 33 ,. , 1 0 / Die = 4 Mortality = 11% 4. %(5) x 3(34). Total no. eggs = 39 Hatch =35 D i e = 3 Mortality = 8% 5. £(6) x <?(33). Total no. eggs = 32 Hatch = 29 M . . . . n o / Die = 3 Mortality = 9% 6. $(4) x <?(33). Total no. eggs = 56 Hatch = 46 ,„ . , . . , a a. Die = 10 Mortality = 18% 7. °-(5) x <?(35). Total no. eggs = 54 Hatch =53 M Die = 1 Mort a l i t y = 2% TOTAL MORTALITY =8.5% oo Trachurus £ x Trachurus <f 1. $(33) x <?(32). Total no. eggs = 88 Hatch =86 Die • 2 Mortality = 2% 2. $(33) x 3(33). Total no. eggs = 52 Hatch = 47 , . D i e _ 5 Mortality = 10% 3. ?(35) x 3(32). Total no. eggs = 82 Hatch =63 M o r t a l i t y = 1 7 % Die =14 J 4. £(32) x 3(33). Total no. eggs = 96 Hatch = 84 . . . . , 0 o / Die = 12 Mortality = 13% 5. ?(34) x 3(35). Total no. eggs = 91 Hatch =88 Die = 3 Mortality = 3% 6. £(33) x 3(31). Total no. eggs = 77 Die C= 2 I 5 6 Mortality = 27% 7. £(32) x 3(33). Hatch = 79 Die = 7 Total no. eggs = 86 Mortality = 8% TOTAL MORTALITY = 11.1% Trachurus Q x Leiurus c r * * 1. £(33) x <?(5). Total no. eggs = 75 Hatch = 74 Die = 1 Mo r t a l i t y = 1% 2. $(33) x 5(5). Total no. eggs = 63 Hatch =59 Die = 4 Mortality = 6% 3. £(35) x 6*(6) . Total no. eggs = 85 Hatch =68 • . 0 f t 0, Die = 17 Mortality = 20% 4. £(32) x 3(5). Total no. eggs = 83 Hatch = 75 . i • . i na/ D £ E _ Q Mortality = 10% 5. ?(34) x 3(4). Total no. eggs = 102 Hatch = 98 D i e _ 4 Mo r t a l i t y = 4% 6. ?(33) x 3(5). Total no. eggs = 68 Hatch = 45 M o r t a l i t = 3 4 o ^ Die = ZJ 7. £(32) x 3(6). Total no. eggs = 94 Hatch = 88 „ . , . . , 0 / n . , Mort a l i t y = 6% Die = 6 TOTAL MORTALITY =11% 00 O Trachurus °_ x Trachurus cr* 1. ¥(31) x 5(35). Hatch = 59 Die = 6 2. ¥(34) x S(33). Hatch = 45 Die = 1 3. $(32) x 3(35). Hatch • 90 Die = 2 4. ¥(33) x 5(33). Hatch = 65 Die = 8 5. ?(32) x 3(33). Hatch = 80 Die = 4 6. ?(32) x 3(31). Hatch = 72 Die = 12 7. 2(33) x 3(32). Hatch = 75 Die = 3 Total no. Mortality Total no. Mortality Total no. Mortality Total no. Mortality Total no. Mortality Total no. Mortality Total no. Mortality eggs = 65 = 9% eggs = 46 = 2% eggs = 92 = 2% eggs = 73 = 11% eggs = 84 = 5% eggs = 87 = 14% eggs = 78 = 4% TOTAL MORTALITY = 6.8% Trachurus ¥ x Leiurus <f 1. 2. 3. 4. 5. 6. 7. 2(31) x 5(5). Hatch = 78 Die = 3 Total no. eggs = 81 Mortality = 4% ¥(34) x 3(6). Total no. eggs = 67 Hatch = 59 .„ . , . . . O Q / Die = 8 Mortality = 12% ¥(32) x 3(5). Total no. eggs = 98 S e * 8 9 0 «°rtality - 8X $(33) x 3(4). Total no. eggs = 61 Hatch =55 M o r t a l i t m 1 0 c / c Die = 6 J ¥(32) x 3(5). Total no. eggs = 76 Hatch = 74 Die = 2 Mortality = 3% ¥(32) x 3(5). Total no. eggs = 112 H * t c h o „ 9 1 Mortality =21% Die =24 * ¥(33) x 3(6). Total no. eggs = 52 Hatch =52 Die = 0 Mo r t a l i t y = 0% TOTAL MORTALITY =9.3% oo L e i u r u s °: x L e i u r u s cf7 1. $ (6) x 3(5). T o t a l n o . eggs = 22 Hatch = 1 8 . , . . n n o / D i e = 4 M o r t a l i t y = 18% 2. ? (5 ) x <?(5). T o t a l n o . eggs = 31 Hatch = 3 1 , i • , D i e = 0 M o r t a l i t y = 0% 3. £ ( 4 ) x <?(6). T o t a l n o . eggs = 27 Hatch = 2 5 D i e = 2 M o r t a l i t y = 7% 4 . ?(4) x 3(5). T o t a l n o . eggs = 33 Hatch = 3 2 D i e = 1 M o r t a l i t y = 3% 5. £ v 5 ) x 3(5). T o t a l n o . eggs = 36 Hatch = 34 D ^ e _ 2 M o r t a l i t y = 5% 6. ?(4) x 3(5). T o t a l n o . eggs = 19 Hatch = 19 D ^ e _ o M o r t a l i t y = 0% 7. S?(4) x 3(4) . T o t a l n o . eggs • 44 Hatch = 3 9 D i e _ 5 M o r t a l i t y = 11% 8. $(4) x 3(4). T o t a l n o . eggs = 78 Hatch = 72 M o r t a l i t y = 8% D i e = 6 TOTAL MORTALITY = 6.8% Le i u r u s °. x H y b r i d o"^  1. $(6) x <?(20). T o t a l n o . eggs = 58 H a t c h = 56 .„ , , . , O D / D i e = 2 M o r t a l i t y = 3% 2. ^(5) x 3(20). T o t a l n o . eggs = 42 Hatch = 39 , , . . _ 0 / D i e = 3 M o r t a l i t y = -7% 3 . £ ( 4 ) x o*(16). T o t a l n o . eggs = 67 Hatch = 66 D i e = 1 M o r t a l i t y = 1% 4 . °-(4) x d'(12). T o t a l n o . eggs = 48 Hatch = 48 D i e = 0 M o r t a l i t y = 0%. 5 . £ ( 5 ) x 3(19). T o t a l n o . eggs = 34 Hatch = 3 3 D i e = 1 M o r t a l i t y = 3% 6. $ (4) x e?(10). T o t a l n o . eggs = 52 Hatch = 49 D i e = 3 M o r t a l i t y = 6% 7. $.(4) x 0^(12). T o t a l n o . eggs = 47 Hatch = 43 D i e = 4 M o r t a l i t y = 8% 8. ?(18) x 3(4). T o t a l n o . eggs = 64 Hatch = 62 M o r t a l i t y = 3 % D i e = 2 TOTAL MORTALITY = 3.8% oo Trachurus J°_ x Trachurus o*"^  1. ¥(32) x c?(33). Total no. eggs = 107 1 Hatch = 104 u ^ ,.^ Die = 3 Mortality = 3% 2. ¥(32) x 5(34). Total no. eggs =92 2 Hatch = 88 A o / Die = 4 Mortality = 4% 3. ¥(33) x 5(32). Total no. eggs = 197 3 ? t G h T o 1 7 9 Mortality = 9% Die =18 4. ¥(34) x 5(33). Total no. eggs = 218 4 Hatch = 211 Die = 7 Mortality = 3% 5. ¥(33) x 3(33). Total no. eggs = 223 5 Hatch = 202 Die = 21 Mortality = 9% 6. ¥(34) x 5(33). Total no. eggs = 184 6 Hatch = 182 , , Die = 2 Mortality = 1% 7. $(35) x 3(33). Total no. eggs = 241 7 Hatch = 230 M . , . . A o / Die = 11 Mortality = 4% 8. ¥(32) x 3(34). Total no. eggs = 212 8 Hatch = 206 M . , . . O o, Die = 6 Mortality = 3% TOTAL MORTALITY = 4.8% Trachurus ° r - x Hybrid 0s9 ¥(32) x 5(21). Hatch =107 Die = 8 -P(32) x 5(11). Hatch 109 Die = 3 3(32) x $(16). Hatch = 72 Die = 0 3(33) x £(15) . Hatch = 52 Die = 4 5(33) x $-(16) . Hatch = 135 Die = 2 Total no. Morta l i t y Total no. Mortality Total no. Morta l i t y Total no. Mortality Total no. Morta l i t y 5(32) x ¥ ( 1 4 ) . Total no. Hatch =149 Die = 4 Mortality 5(33) x ¥(20). Hatch =137 Die m 7 Total no. Mor t a l i t y 3(34) x ¥(18). Total no. Hatch = 86 Die = 1 Mor t a l i t y TOTAL MORTALITY = 3.3% eggs = 7% eggs = 3% eggs = 0% eggs = 7% eggs = 1% eggs = 3% eggs = 5% eggs = 1% = 115 = 112 = 72 = 56 = 137 = 153 = 144 = 87 CD H y b r i d ¥ x L e i u r u s <f 1. $(20) x 3(4)'., T o t a l n o . eggs = 44 H a t c h = 4 4 „ . , • . n o / D i e = 0 M o r t a l i t y = 0% 2. ? ( I 9 ) x 5 ( 5 ) . T o t a l n o . eggs = 56 H a t c h = 53 M o r t a l i t y = 5% D i e = 3; J 3. ¥ ( 1 5 ) x 3 ( 5 ) . T o t a l n o . eggs = 37 H a t c h = 36 „„ . , . . D i e = 1 M o r t a l i t y = 3% TOTAL, MORTALITY =2 .9% H y b r i d ¥ x H y b r i d cf7 1. ¥ ( 2 0 ) x 6^(15). T o t a l n o . eggs Hatch = 62 D i e = 5 M o r t a l i t y = 7% £ ( 1 9 ) x 3(16) . Hatch = 67 D i e = 7 2(15) x c?(21) . Hatch = 81 D i e = 2 T o t a l n o . eggs M o r t a l i t y = 9% T o t a l n o . eggs M o r t a l i t y = 2% = 67 = 74 = 83 TOTAL MORTALITY = 6.2% co 85 provided but hatching success within these groups compares favourably with survival of other homogametic crosses (Table VII). Again the results do not indicate genetic incompatibility or hybrid breakdown. Survival of Fry Mort a l i t y i n F^ hybrids was r e l a t i v e l y high during the f i r s t two weeks afte r hatching, as i t was i n a l l classes of laboratory reared f i s h (Table VIII). A f t e r two weeks f i s h r a r e l y died, but at one month a protozoan parasite, Ichthyophthirius, was introduced into the nursery from wild caught f i s h . Fry were treated with standard s a l t water remedy (Davis, 1961) which checked spread of the disease and cured many of the infected i n d i v i d u a l s . M ortality at the end of three months was comparable between F^ hybrid classes and controls. There was complete mortality i n the one backcross class, which occurred at 33 days and persisted for a four day period. It seemed improbable that disease or laboratory conditions were responsible because a l l f i s h were held i n a common supply of f r e e l y c i r c u l a t i n g water, and there was no comparable mortality i n other groups of f r y . At the time, hybrid breakdown was suspected and both backcross classes were made the following year. Conditions for rearing f r y were the same i n 1965 except that at two months a l l f r y were transferred from baskets to 40 gallon aquaria provided with f i l t e r s and aeration. In a n t i c i p a t i o n of the developmental mortality T a b l e V I I I . S u r v i v a l o f l a b o r a t o r y r e a r e d h y b r i d s w i t h c o n t r o l s p r e s e n t e d i n l e f t hand columns . SURVIVAL TO 3 MONTHS C o n s p e c i f i c H e t e r o s p e c i f i c L e i u r u s x L e i u r u s L e i u r u s £ x T r a c h u r u s cf T o t a l h a t c h l i n g s = 600 M o r t a l i t y a t 2 weeks = 413(31%) M o r t a l i t y a t 1 month = 397(34%) F i s h r e m a i n i n g a f t e r 3 months = 184 - i . e . 69% m o r t a l i t y T o t a l h a t c h l i n g s = 596 M o r t a l i t y a t 2 weeks = 438(27%) M o r t a l i t y a t 1 month = 415(30%) F i s h r e m a i n i n g a f t e r 3 months = 75(85%) T r a c h u r u s x T r a c h u r u s T r a c h u r u s £ x L e i u r u s cf* T o t a l h a t c h l i n g s = 994 M o r t a l i t y a t 2 weeks = 674(32%) M o r t a l i t y a t 1 month = 624(36% F i s h r e m a i n i n g a f t e r 3 months = 2 T o t a l h a t c h l i n g s - 1006 M o r t a l i t y a t 2 weeks = 632(37%) M o r t a l i t y a t 1 month = 591(41%) F i s h r e m a i n i n g a f t e r 3 months = 109(89%) H y b r i d % x L e i u r u s (f T o t a l h a t c h l i n g s = 122 M o r t a l i t y a t 2 weeks = 92 M o r t a l i t y a t 1 month = 100% No s u r v i v o r s OD 0) SURVIVAL TO 4 MONTHS C o n s p e c i f i c H e t e r o s p e c i f i c L e i u r u s x L e i u r u s cf L e i u r u s 5- x H y b r i d cf7 T o t a l h a t c h l i n g s = 2 1 8 M o r t a l i t y a t 2 weeks = 24%(165*) M o r t a l i t y a t 1 month = 27%(157*) M o r t a l i t y a t 4 months = 39%(132*) T o t a l h a t c h l i n g s = 243 M o r t a l i t y a t 2 weeks = 18%(199*) M o r t a l i t y a t 1 month = 25%(182*) M o r t a l i t y a t 4 months = 33%(162*) L e i u r u s cf x H y b r i d °-T o t a l h a t c h l i n g s = 61 M o r t a l i t y a t 2 weeks = 23%(47*) M o r t a l i t y a t 1 month = 29%(42*) M o r t a l i t y a t 4 months = 36%(39*) T r a c h u r u s cf x H y b r i d •?• T o t a l h a t c h l i n g s - 136 M o r t a l i t y a t 2 weeks = 16%(114*) M o r t a l i t y a t 1 month = 21%(107*) M o r t a l i t y a t 4 months = 32%(93*) T r a c h u r u s 2- x H y b r i d cf T o t a l h a t c h l i n g s = 74 M o r t a l i t y a t 2 weeks = 22%(58*) M o r t a l i t y a t 1 month = 30%(52*) M o r t a l i t y a t 4 months = 34%(49*) * = s u r v i v o r s 87 at 33 days, i n 1965 rep l i c a t e s of backcrosses were made at two week in t e r v a l s so that i f death were a r e s u l t of hybrid breakdown there should be a two week i n t e r v a l among repl i c a t e s when death occurred. The expected mortality did not eventuate (Table VIII). Indeed, at the end of four months sur v i v a l of backcrosses was, i f anything, s l i g h t l y better than su r v i v a l i n the r a c i a l crosses. No explanation can be offered f o r the discrepancy i n resu l t s , but i t i s important to r e c a l l that only one backcross combination was made i n 1964 while a l l four were made i n 1965 producing larger and more complete samples the second year. Despite good s u r v i v a l and seemingly normal and vigorous hybrid offspring, f i v e F-^ ' s and three backcrosses were abnormal. The deformities i n a l l the abnormal hybrids were a l i k e and consisted of the following: a. no swim bladder b. i n t e s t i n e without the normal posterior loop, and consisting of a str a i g h t tube from mouth to anus c. abnormally large eyes d. dwarfed size, about h a l f that of normal hybrids e. very th i n and bow-shaped bodies. These indivi d u a l s swam by f l u t t e r i n g the pectoral f i n s , and this along with an upset i n t h e i r density r e s u l t i n g from the absence of a swim bladder produced very e r r a t i c and j i t t e r y motion when swimming. They spent most of the time sheltered i n algae on the surface and i f they were forced out they sank to the bottom and could return to the surface only with much e f f o r t . Hubbs 88 (192 7) r e p o r t e d s i m i l a r a b n o r m a l i t i e s i n p a r a s i t i z e d f i s h , and s i n c e some eggs had been exposed to fungus a t a l a t e stage of development i t i s not p o s s i b l e to a t t r i b u t e the a b n o r m a l i t i e s e i t h e r to an i n c i p i e n t h y b r i d breakdown or to p a r a s i t i s m . I f the cause were the former i t e x i s t s a t an extremely low l e v e l . A t f o u r months males had become a g g r e s s i v e and had a b l u e i r i s , s u g g e s t i n g they were approaching s e x u a l m a t u r i t y but t h i s ensued a t a c o n s t a n t p h o t o p e r i o d o f 16 hours and a r e l a t i v e l y h i g h temperature o f 17° to 19°C (but see Baggerman 1957). G r a v i d females were not found but n e i t h e r was i t p o s s i b l e to induce g r a v i d i t y i n p a r t i a l l y mature females ca p t u r e d i n the w i l d and kept i n the l a b o r a t o r y . H y b r i d F e c u n d i t y A measure of h y b r i d f e c u n d i t y was d e r i v e d from counts of mature eggs i n h y b r i d s as compared w i t h counts from the two r a c e s . Once i t has been e s t a b l i s h e d t h a t s t e r i l i t y b a r r i e r s are not p r e s e n t such counts of eggs can p r o v i d e i n f o r m a t i o n about f e c u n d i t y o f h y b r i d s , although they give no i n f o r m a t i o n c o n c e r n i n g the number o f c l u t c h e s h y b r i d s may produce. The r e s u l t s (Table IX) show t h a t female h y b r i d s are i n t e r m e d i a t e i n f e c u n d i t y between the two races producing more eggs than l e i u r u s but l e s s than t r a c h u r u s . T h i s r e s u l t i s not s u r p r i s i n g s i n c e egg p r o d u c t i o n i n each race i s l a r g e l y a f u n c t i o n of s i z e , and s i n c e h y b r i d s are i n t e r m e d i a t e between the races i n s i z e . T a b l e I X . Numbers of mature eggs f o r h y b r i d s and f o r each r a c e . Sample s i z e o f 40 f o r each race and f o r h y b r i d s . L e i u r u s H y b r i d s T r a c h u r u s N o . No . No. No. No. N o . Length eggs Length eggs Length eggs Length eggs Length eggs Length eggs 4 .8 cm 112 4 . 7 cm 96 5.4 cm 143 5.8 cm 217 5 .4 cm 210 5 .6 cm 225 4 . 9 115 3 .9 83 4 . 7 106 6 229 5.2 157 5 .8 257 5 .2 148 5 123 4 .7 142 5.9 284 5 .8 227 6.1 231 5.3 120 5.2 205 5 .1 207 5 .8 236 6 239 6.2 284 4 72 5.2 170 6 236 5 .9 249 5 .9 286 5 .7 226 3 .6 75 5 172 5.5 186 5 .9 247 6 223 4 . 4 101 4 . 4 124 6.3 296 6 204 5 .8 253 5 .8 191 4 93 4 .2 71 5 .9 226 5 .7 217 5 .8 171 5 .7 267 4 .2 76 5 .4 229 4 .8 112 5.4 227 6 205 5 .9 256 4 88 5 .8 234 5 .1 132 5 .6 231 5 .7 228 5 .9 284 4 . 4 123 7.5 229 5.3 126 6.1 239 5 .9 247 6 257 4 .3 60 3.4 51 4 .6 93 5 .6 217 5 .7 243 5 .8 239 4 . 1 56 3 .4 49 4 83 6 227 6.3 259 5.2 162 4 .3 69 4 .5 76 5.3 123 5.9 237 6.4 271 5 .4 221 3.3 71 4 .2 79 4 .9 126 5.4 152 5 .7 213 5 .9 253 4 . 6 84 4 . 1 57 4 .8 123 4 .8 118 6 228 5 .8 248 5.2 116 3 .8 52 6 224 4 . 7 150 6.1 257 6.2 271 5 .1 124 4 .5 70 5 .7 201 5.2 216 5 .9 283 6.5 292 4 .5 92 4 67 5 .4 212 5.9 224 5 . 7 219 5 .6 209 4 . 7 97 4 .3 76 5.2 169 6.1 283 5 .9 266 6 232 X = 105 X = 190 X = 241 90 Hybrid I n f e r i o r i t y It i s exceedingly d i f f i c u l t to gain information pertaining to the s e l e c t i v e disadvantage of hybrids r e l a t i v e to parental types, f i r s t because the s e l e c t i v e agents operating against hybrids may be very low i n i n t e n s i t y or very subtle i n action, and second because any negative evidence attained i n the laboratory of the absence of s e l e c t i o n against hybrids i s meaningless since the s e l e c t i v e agents themselves may have been removed. In early September of 1964 the entire one mile hybrid zone was sampled and percentages of hybrids and races estimated. The procedure was repeated again i n early June of 1965 thus giving estimates of r e l a t i v e numbers of hybrids i n autumn af t e r reproduction ceased, and again i n late spring while young of the year could be i d e n t i f i e d and excluded from counts. I f hybrids were at a s e l e c t i v e disadvantage r e l a t i v e to either race, one should be able to detect a decrease i n proportion of hybrids over this nine month lapse. For September census figures were: 217 leiu r u s . 82 hybrids, and 14 trachurus. Estimates for June were: 246 leiurus , 73 hybrids, and 12 trachurus. This measure, rough though i t i s , gives no i n d i c a t i o n that hybrids suffer any manifest disadvantage as compared to parental types, and t h i s together with an abundance of backcrosses i n the hybrid zone indicates that either hybrids are not at a s e l e c t i v e disadvantage or that they are at a very low s e l e c t i v e disadvantage i n the hybrid zone. 91 DISCUSSION ISOLATING MECHANISMS AND HYBRIDIZATION Iso l a t i n g Mechanisms A morphological analysis and observations i n the f i e l d provide firm circumstantial evidence that leiurus and trachurus f r e e l y interbreed i f they come together, producing vigorous and f e r t i l e hybrids. Investigations i n the L i t t l e Campbell River and Bonsall Creek reveal that the two races are l a r g e l y segregated, but where they cohabit presumed hybrids and backcrosses are p l e n t i f u l . And again when leiurus i s trans-planted into a breeding population of trachurus numerous hybrids a r i s e . This evidence would seem to b e l i t t l e the importance of behavioural or genetic obstacles to hy b r i d i z a t i o n . The v i r t u a l absence of ethological and postmating i s o l a t i n g mechanisms (excluding hybrid i n f e r i o r i t y ) i s confirmed by experimental analysis i n the laboratory. Behavioural patterns that function as i s o l a t i n g mechanisms could not be detected, and mating between races i s random i n preference t e s t s . Hybrids were found to successfully complete courtship and parental a c t i v i t i e s . S t e r i l i t y b a r r i e r s are apparently not present nor i s genetic incompatibility, for i f the few abnormal hybrids that were found i n the laboratory are a r e s u l t of incompatibility (and not p a r a s i t i s m ) , i t furnishes a very i n e f f e c t i v e block to gene flow. Most of the hybrids are vigorous and no decrease i n hatching success or s u r v i v a l of o f f s p r i n g could be found. 92 F i e l d and l a b o r a t o r y d a t a g a t h e r e d i n t h i s r e s e a r c h demonstrate t h a t r e p r o d u c t i v e i s o l a t i o n s p r i n g s p r i m a r i l y f rom e c o l o g i c a l i s o l a t i o n and to a l e s s e r degree from s e a s o n a l i s o l a t i o n . S e a s o n a l i s o l a t i o n i s p a r t i a l s i n c e the two r a c e s b r e e d a t the same time f o r o n l y one month out of our t h a t each i s b r e e d i n g . However, t h i s i s o l a t i o n i s f u r t h e r d i m i n i s h e d inasmuch as the numbers o f l e i u r u s t h a t are b r e e d i n g have d e c r e a s e d when t r a c h u r u s move i n t o the s t ream to b r e e d , and c o n v e r s e l y , when l e i u r u s i s a t the peak o f b r e e d i n g season t h e r e are o n l y v e r y few e a r l y spawning t r a c h u r u s p r e s e n t i n the s t r e a m . E a r l y spawning t r a c h u r u s makes a major c o n t r i b u t i o n to h y b r i d i z a t i o n i n the L i t t l e C a m p b e l l R i v e r . They a r r i v e i n the s t ream w h i l e the g r e a t e s t numbers o f l e i u r u s are b r e e d i n g , move f a r t h e r upstream than the p r i n c i p a l spawning m i g r a t i o n , and s e t t l e i n an i n t e r m i n g l e d h a b i t a t where s p a r s e b u t permanent p o p u l a t i o n s o f l e i u r u s o c c u r . H y b r i d swarms have d e v e l o p e d a t these s i t e s . T h i s s i t u a t i o n c o n t r a s t s w i t h t h a t i n B o n s a l l Creek where h y b r i d i z a t i o n r e s u l t s not f rom e a r l y m i g r a n t s b u t f rom c o n t i g u o u s h a b i t a t s and, t h e r e f o r e , c o n t i g u o u s p o p u l a t i o n s o f the two r a c e s . E c o l o g i c a l i s o l a t i o n s e r v e s v e r y p o w e r f u l l y to p r e v e n t h y b r i d i z a t i o n . The r a c e s are i n t i m a t e l y a s s o c i a t e d w i t h d i f f e r e n t h a b i t a t s , which are l a r g e l y s e p a r a t e , and t h i s e x c l u s i o n o f h a b i t a t s p r e v e n t s b i o t i c sympatry (Grant , 1963) . L e i u r u s i n h a b i t s q u i e t and s t a n d i n g backwaters o r ponds w i t h a muddy bot tom, dense s t a n d s o f a q u a t i c v e g e t a t i o n , and u s u a l l y , t e a - c o l o u r e d w a t e r . T r a c h u r u s , on the o t h e r hand, b r e e d s i n a 93 habitat characterized by a sandy bottom and a mild current, beds of recumbent vegetation, usually Elodea or Myriophyllum, and clear water. These conditions held i n a l l streams examined, but Bonsall Creek was e s p e c i a l l y i n s t r u c t i v e . In that stream a switch i n habitats occurs within 200 feet, and with i t there i s a complete s h i f t i n the two races. Leiurus i s an abundant f i s h upstream i n i t s t y p i c a l habitat, and trachurus i s numberous downstream i n i t s t y p i c a l habitat. In the b r i e f intermediate zone trachurus i s present i n mid-stream where there i s a sand bottom, mild current, and stands of Elodea, while along the margins of the stream leiurus occurs over a mud bottom with dense emergent and submerged aquatic vegetation, and s t i l l water. More evidence favouring ecological i s o l a t i o n comes from dispersal and transfer experiments. When leiurus i s moved from one portion of i t s habitat to another i t tends to sort out into quiet backwaters with dense vegetation along the margins of the stream. Large numbers of leiurus transferred to the lower part of the stream i n winter - the summer breeding location of trachurus - rapi d l y move out of the area. At that time of the year there i s no re t r e a t from swift current, and vegetation i s not present. Such a h o s t i l e environment would pr o h i b i t leiurus from establishing; permanent populations i n the lower reaches of the stream, and thus prevent interbreeding between races. However, i f leiurus i s transferred to the same l o c a l i t y i n the summer i t establishes residence while the current i s less and s t i l l water can be found along the margins of the 9 4 stream, and Elodea covers the bottom. Once again leiurus vanishes from the area i n winter as current increases and vegetation disappears. Conversely, when trachurus i s planted i n a leiurus habitat th e i r numbers diminish over one month u n t i l none remains. There i s evidence suggesting a'downstream migration from the area, but the loss of bright breeding colours, th e i r t h i n and emaciated appearance, and the many dead individuals found, a l l indicate that trachurus i s i l l - a d a p t e d to the habitat. For the month they were present i n the upper reaches they congregated over r e l a t i v e l y swift r i f f l e s with gravel bottoms. Results of preference tests i n the laboratory substantiate evidence from the f i e l d for e c o l o g i c a l i s o l a t i o n . Leiurus has a strong preference for nesting on a mud substrate, and trachurus selects sand as a nesting location. Leiurus demonstrates a preference for nesting among Oenanthe (an exposed location from the viewpoint of the f i s h but often with a dense canopy of leaves above), and trachurus a preference for nesting among Elodea (secluded at the nest s i t e ) . Even though both races choose to nest i n standing water trachurus spends much more time i n the h a l f of the tank with current than does l e i u r u s . Results of preference tests were not always exclusive between races, for even though leiurus i s associated with tea-coloured water i n the wild i t shows a preference for clear water from Station N. In addition, evidence from morphology and behaviour indicates that the two races are thoroughly adapted to d i f f e r e n t 95 habitats. The terete shape found i n trachurus i s a well known adaptation to l i f e i n current, and the deeper shape of leiurus an adaptation to l i f e i n standing water (Hubbs, 1940). It has been demonstrated that d i s t i n c t trophic adaptations e x i s t between the two races, trachurus possessing many g i l l rakers with a long and fine architecture, and leiurus with few g i l l rakers that are short and thick. An analysis of stomach contents" suggest that leiurus feeds predominately on bottom dwelling organisms, and that trachurus feeds on planktonic or suspended organisms. Observations on feeding behaviour i n 40 gallon aquaria indicate that leiurus feeds mostly on larger p a r t i c l e s of food that have s e t t l e d to the bottom, and trachurus feeds l a r g e l y on suspended p a r t i c l e s . This was e s p e c i a l l y noticeable when f i s h were fed brine shrimp. Leiurus was often observed i n the stream carrying on what I assume were feeding a c t i v i t i e s , and these were very c h a r a c t e r i s t i c . The f i s h would swim slowly along just above the bottom and eventually come to an abrupt stop with the body t i l t e d at about a 45° angle, linger for a moment and dart forward into the mud causing a small cloud to r i s e . A f t e r backing away i t could be seen making a mulling motion with the mouth. This a c t i v i t y , or others that seemed associated with feeding, was never discerned i n trachurus. In both the morphological and behavioural aspect of trophic s p e c i a l i z a t i o n then, the two races are well adapted to the i r habitats one for gathering food from the bottom i n quiet, muddy, and vegetated habitats, and the other for taking suspended organisms i n either a stream or i n the sea where trachurus lead a pelagic 96 existence. Other differences concerning location of nesting s i t e s may well be adaptations to l i f e i n standing or flowing water, which would further function to i s o l a t e the races e c o l o g i c a l l y . Leiurus i n the wild and i n aquaria during preference tests choose open nesting locations. In s t i l l water, where they dwell, there would be no s e l e c t i v e pressure favouring a protection of nests and f r y from current that may sweep them away from the parent. But there may be s e l e c t i o n favouring an exposed nesting s i t e where a male i n a dense population : (and populations of leiurus are usually dense) would have a better opportunity to encounter a female, or the female to locate a nesting male. Evidence from the c o l l e c t i o n of nests i n the wild and from preference tests demonstrates that trachurus places i t s nest within the shelter of dense vegetation. It also nests where current p r e v a i l s . In that habitat s e l e c t i o n may be the other way around, as i t were. There may then be a sel e c t i v e premium fo r placing nests where they are protected from current that could sweep f r y away from the parent, and since the vegetation present i n that habitat i s recumbent the males are more exposed to females for courtship. It was observed that trachurus i s a more shy and r e t i r i n g f i s h than l e i u r u s . It i s more d i f f i c u l t to induce trachurus to breed i n aquaria and i t spends more time hidden from view, while i n the wild i t i s equally shy and their nests more d i f f i c u l t to locate. A s l i g h t movement from an observer on the bank of the stream would send trachurus darting into 97 a b e d o f E l o d e a . T h i s c o n t r a s t s w i t h l e i u r u s w h i c h i s e a s i l y -o b s e r v e d and l e s s s h y . A g a i n these d i f f e r e n c e s i n b e h a v i o u r may be a d a p t a t i o n s to the h a b i t a t s f r e q u e n t e d by each r a c e ; l e i u r u s i n a h a b i t a t w i t h an o b s c u r i n g mud b a c k g r o u n d u s u a l l y w i t h a dense canopy o f v e g e t a t i o n above, t r a c h u r u s i n an exposed h a b i t a t w i t h a l i g h t background o f sand and o n l y recumbent v e g e t a t i o n w i t h no canopy o f l e a v e s a b o v e . Most o f the d i v e r g e n c e t h a t has ensued between the r a c e s i s a r e s u l t o f s e l e c t i o n and a d a p t a t i o n i n two c o n t r a s t i n g e n v i r o n m e n t s , p r o d u c i n g m a n i f o l d e f f e c t s t h a t a re e x p r e s s e d i n the morphology, h a b i t a t p r e f e r e n c e , and b e h a v i o u r . The outcome o f t h i s s e l e c t i o n has been e c o l o g i c a l i s o l a t i o n , w h i c h has a c o n s i d e r a b l e g e n e t i c b a s i s . Most o f the m o r p h o l o g i c a l d i f f e r e n c e s t h a t d i s t i n g u i s h the r a c e s are under c o n t r o l o f p o l y g e n e s , and i t may be e x p e c t e d t h a t o t h e r d i f f e r e n c e s , as h a b i t a t p r e f e r e n c e , b e h a v i o u r , and s e a s o n a l i s o l a t i o n , a l s o r e p r e s e n t c o n s i d e r a b l e g e n e t i c d i f f e r e n t i a t i o n . I t i s then somewhat s u r p r i s i n g t h a t i s o l a t i n g mechanisms so i m p o r t a n t i n o t h e r a n i m a l s , e t h o l o g i c a l and p o s t m a t i n g , have not d e v e l o p e d as an i n c i d e n t a l p r o d u c t . F o r as Mayr (1963) makes c l e a r the genotype i s a c o a d a p t e d and i n t e g r a t e d u n i t , and each i s o l a t i n g mechanism has an e x t e n s i v e g e n e t i c b a s i s . The H y b r i d Zone H y b r i d s c o n s t i t u t e about 21% of the s t i c k l e b a c k s i n the h y b r i d zone , b u t they may be as abundant as 46% a t some l o c a l i t i e s ( S t a t i o n s M and M') and a t these s i t e s the 98 proportion of trachurus, hybrids, and leiurus i s 1:8:10. Early spawning trachurus are scarce and w i l l perpetuate h y b r i d i z a t i o n by outcrossing with the more abundant l e i u r u s . Bagkcrossing i s most frequent to leiurus, for which there are probably several reasons. Leiurus i s much more abundant than trachurus, so that once F i 1 s are present t h e i r chances of encountering leiurus are greatest. Furthermore, leiurus i s a permanent resident i n the hybrid zone and this provides considerable opportunity for backcrossing to that race before trachurus arrives (Figure 12). F i n a l l y , the seasonal i s o l a t i o n between sexes of hybrids diminishes the chances for an F2 generation. But, as the hybrid index shows, there i s some backcrossing to trachurus, and probably occasional interbreeding among hybrids. An aspect of hybrid zones i n sticklebacks that i s d i f f i c u l t to understand i s t h e i r narrowness; why has swamping not occurred? Seasonal i s o l a t i o n reduces the opportunity for h y b r i d i z a t i o n and ec o l o g i c a l i s o l a t i o n very e f f e c t i v e l y minimizes interbreeding but, nonetheless, where these mechanisms have f a i l e d why has swamping not ensured i n the absence of behavioural or postmating b a r r i e r s ? It i s clear that the duration of hybrid zones cannot be established, but they are repeated many times i n d i f f e r e n t streams. Despite the f a c t that with random mating panmixia could occur within one generation, hybrid zones remain narrow and the two races remain d i s t i n c t and i s o l a t e d . Several factors seem to be involved: 1. Evidence that leiurus i s a very sedentary f i s h has come from several l i n e s ; from p a r a s i t i z e d 99 populations that are l o c a l i z e d , from the c l i n e i n plate numbers i n leiurus, the r e s t r i c t i o n of hybrids to points of hybridization (where the two races coexist), and from dispersal experiments. A sedentary habit reduces interchange between hybrids and either race. Fryer (1959) has demonstrated that parental care i n c i c h l i d s serves to r e s t r i c t dispersal of offspring, which become habituated to the l o c a l surroundings as a r e s u l t of t h e i r late emancipation from parents. The intense parental care i n sticklebacks may well function to reduce dispersal as i t does i n c i c h l i d s . The two races have strong habitat preferences and s e l e c t habitats with d i f f e r e n t environmental c h a r a c t e r i s t i c s . The hybrid zone, however, has c h a r a c t e r i s t i c s that are intermediate between the habitats of e i t h e r race, and the d r a s t i c reduction i n density of both races i n that zone t e s t i f i e s that i t i s less desirable. Hybrids have attributes that are intermediate between the races and i t i s probable they have a preference for and s e l e c t the intermediate zone, but avoid leiurus and trachurus habitats that are j u s t up and downstream. The nonrandom d i s t r i b u t i o n found i n the hybrid classes provides evidence supporting t h i s explanation (there was no evidence 100 that trachurus was nonrandomly d i s t r i b u t e d ) . As discussed e a r l i e r the two races are well adapted to the divergent habitats that each l i v e s i n and i l l adapted to the habitat of the other. This i s apparent i n the morphology of the food gathering apparatus, body shape, feeding behaviour, and nesting behaviour. But hybrids are intermediate i n number and structure of t h e i r g i l l rakers, and i t i s not u n l i k e l y that they are also intermediate i n feeding behaviour. Presumably they are not as well adapted i n either habitat as the two races are i n t h e i r own habitat. For example, hybrids are probably poorly adapted to c o l l e c t bottom dwelling organisms or planktonic and d r i f t i n g organisms, both because t h e i r feeding apparatus i s intermediate and because th e i r behaviour i s adjusted to c o l l e c t neither. With intermediate body shapes they are less adapted to standing and to flowing water. If they are intermediate i n nesting behaviour, such an upset could cause males to place nests i n current that would sweep the f r y away, or i n dense vegetation where there i s a s e l e c t i v e premium for males that have exposed and conspicuous s i t e s . There are undoubtedly many other more subtle, but equally important, adaptive t r a i t s the races possess, and many of these must impose a sele c t i v e disadvantage 101 against hybrids. Furthermore, there are dense populations of leiurus upstream and dense populations of trachurus below the hybrid zone. Thus, i t i s reasonable to suppose that hybrids are at a considerable s e l e c t i v e disadvantage on both sides beyond the hybrid zone, for there they are less well adapted and must compete with well adapted populations i n a saturated habitat. Selection against hybrids could not be found, however, inside the hybrid zone, nor would i t be expected i n view of the foregoing discussion. Inside the hybrid zone where the habitat i s intermediate both races are at a selec t i v e disadvantage since they are less well adapted and, hence, t h e i r numbers reduced. But hybrids with t h e i r intermediate t r a i t s may not be at a sele c t i v e disadvantage r e l a t i v e to either race because the habitat i s intermediate and because competition would be greatly reduced. The circumstances explained above could e a s i l y give r i s e to a stable hybrid zone i n which neither swamping nor reinforcement could take place for considerable time. Behavioural and postmating i s o l a t i n g mechanisms (excluding hybrid i n f e r i o r i t y ) have not accompanied divergence between the two races so that where they come into contact there i s no means to prevent interbreeding. On the other hand ecol o g i c a l i s o l a t i n g mechanisms are well developed and contact between races arises only where habitats are contiguous. Once contact i s established the extent of h y b r i d i z a t i o n w i l l depend upon the size of the intermediate 102 habitat where hybrids are not at a sel e c t i v e disadvantage. If the intermediate zone i s r e l a t i v e l y broad as i t i s i n the L i t t l e Campbell River (1 mile) h y b r i d i z a t i o n may be extensive, but i f i t i s narrow as i t i s i n Bonsall Creek (200 feet) hybridization w i l l be severely r e s t r i c t e d . In either instance the hybrid zone cannot broaden, nor swamping take place, becuase of the sel e c t i v e disadvantage outside the zone. S t i l l h y b r i d i z a t i o n continues inside the hybrid zone because there i s no i s o l a t i n g mechanism to prevent i t and because hybrids are not at a disadvantage there. Even i f reinforcement could occur i n a hybrid zone where hybrids are not at a disadvantage, the reinforced genotypes that developed i n the zone would very l i k e l y be swamped by the v a s t l y greater number of genotypes of either race that occur outside the zone. This complex of factors together with s e l e c t i v e disadvantage against hybrids absent inside the zone but present outside the zone could produce s t a b i l i t y over long periods. According to the concept of reinforcement, where individuals of two species (or divergent populations) overlap, those individuals that have poorly developed i s o l a t i n g mechanisms w i l l be most prone to hybridize, and w i l l produce hybrids that are at a s e l e c t i v e disadvantage. Individuals that have better developed i s o l a t i n g mechanisms w i l l be l e a s t prone to hybridize and w i l l therefore leave more o f f s p r i n g . Thus, natural s e l e c t i o n w i l l perfect i s o l a t i n g mechanisms because genotypes that hybridize w i l l be l o s t from either species (or population), and eventually h y b r i d i z a t i o n w i l l no longer occur. Moore (1957) has c r i t i c i z e d 103 this concept, pointing out that reinforcement of i s o l a t i n g mechanisms i s an ad hoc mechanism; that i s , reinforcement of i s o l a t i n g mechanisms i s only of s e l e c t i v e value inside the hybrid zone because only within the zone are genotypes that hybridize at a s e l e c t i v e disadvantage. Outside the hybrid zone such reinforced genotypes would be s e l e c t i v e l y neutral or even disadvantageous. The l a t t e r argument may apply to i s o l a t i n g mechanisms such as ethological i s o l a t i o n , preventing t h e i r spread beyond the hybrid zone; that i s , providing they could arise despite swamping from the dense populations of unreinforced genotypes that occur outside the zone. However, Moore's c r i t i c i s m i s concerned only with the spread of perfected i s o l a t i n g mechanisms outside the hybrid zone, and i t seems u n l i k e l y that t h i s c r i t i c i s m applies to e c o l o g i c a l i s o l a t i n g mechanisms. This i s so for the following reasons: Ecological i s o l a t i o n develops outside the hybrid zone i n response to s e l e c t i o n and adaptation to two diverse environments, and s e l e c t i o n would be expected to operate constantly to perfect adaptation and the consequent ecological i s o l a t i o n . Those individuals within either race that have poorly perfected e c o l o g i c a l i s o l a t i n g mechanisms w i l l be more prone to move into the hybrid zone than individuals with well developed i s o l a t i n g mechanisms. Once i n the hybrid zone the former are at a severe s e l e c t i v e disadvantage. F i r s t , they are poorly adapted to the intermediate habitat i n the zone, and second they w i l l probably hybridize and t h e i r genotypes w i l l be l o s t from that race because t h e i r hybrid o f f s p r i n g cannot get out of the hybrid zone, or i f they did they would be severely selected against. Thus, there i s no concern to have reinforced i s o l a t i n g mechanisms spread beyond the hybrid zone. On the contrary, those individuals with poorly developed ecological i s o l a t i o n move into the hybrid zone arid are selected against. Individuals with better developed i s o l a t i n g mechanisms w i l l increase, and the r e s u l t w i l l be reinforcement and s t r i c t habitat s e l e c t i o n . But no matter how well developed eco l o g i c a l i s o l a t i o n becomes i t would seem to be always less than perfect. In sticklebacks e c o l o g i c a l i s o l a t i o n might become so perfected, as a r e s u l t of reinforcement, that s t r i c t habitat s e l e c t i o n and adaptation would ensure. Even i f t h i s occurred, however, habitats may l i e side by side as they do i n Bonsall Creek, and straying of no more than feet (or inches) might r e s u l t i n h y b r i d i z a t i o n . Trautman (1948) reports that two species of f i s h (Schilbeodes) are normally well separated by e c o l o g i c a l i s o l a t i o n , but habitats of each species may be within inches of one another, r e s u l t i n g i n h y b r i d i z a t i o n . Many sim i l a r example are known i n f i s h (Trautman, 1957; Hubbs, 1961; for reviews). Reference to Mayr's c l a s s i f i c a t i o n of i s o l a t i n g mechanisms (Introduction) shows that he l i s t s e c o l o g i c a l i s o l a t i o n as a premating mechanism and hybrid i n f e r i o r i t y as a postulating mechanism. Mayr (1963) states "There i s a fundamental difference between the two types: premating mechanisms prevent the wastage of gametes and so are highly susceptible to improvement by natural s e l e c t i o n : postmating 105 mechanisms do not prevent wastage of gametes and t h e i r improvement by natural s e l e c t i o n i s d i f f i c u l t and i n d i r e c t " . This implies that the mode of operation of natural s e l e c t i o n w i l l be very d i f f e r e n t for pre and postmating i s o l a t i n g mechanisms. It i s obvious, however, that i n sticklebacks there i s no separation between some pre and postmating i s o l a t i n g mechanisms. Ecological i s o l a t i o n (a premating mechanism) produces a postmating i s o l a t i n g mechanism (hybrid i n f e r i o r i t y ) . The two cannot be separated. Each race i s well adapted to a d i f f e r e n t habitat, and t h i s adaptation (ecological i s o l a t i o n ) produces poorly adapted hybrids (hybrid i n f e r i o r i t y ) because they are intermediate. There are numerous examples of hybrid zones where i t seems that ecological i s o l a t i o n and hybrid i n f e r i o r i t y go hand i n hand (Dixon, 1955; Ingles and Biglione, 1951; Hubbs, 1961; Meise, 1928; Mayr and G i l l i a r d , 1952; Hubbs and M i l l e r , 1943). I t i s doubtful, i n a l l cases of hybrid zones, that e c o l o g i c a l i s o l a t i o n and hybrid i n f e r i o r i t y can be separated, and hence, whether a d i s t i n c t i o n can be made between the two modes of operation of natural s e l e c t i o n that Mayr implies. SPECIATION I t i s the opinion of the writer that there i s a need to consider the subject of speciation with special caution, for the following reasons. Speciation i s a r e l a t i v e l y youthful f i e l d i n biology (Mayr, 1957), one i n which our evidence i s i n d i r e c t and based on inference. The second point i s best expressed i n 106 the words of Mayr (1959) who states "The more I study evolution the more I am impressed by the uniqueness, by the u n p r e d i c t a b i l i t y , and by the unrepeatability of evolutionary events.", and goes on to suggest that i t may be a mistake to apply a generalizing technique l i k e mathematics to a f i e l d of events so unique as evolution. In view of the youth of the study of speciation/ the i n d i r e c t nature of the evidence, and the uniqueness of evolutionary events, the time has not come when we can generalize from a few examples so far as to exclaim that a l l speciation occurs i n this or that manner. Herein l i e s the need for sp e c i a l caution, for there i s a tendency to assume the conclusion for a given case of speciation; that i t i s geographic. Geographic speciation has been so thoroughly expounded upon (see Mayr 1963 for a review) that there i s no need to treat i t i n d e t a i l here. It i s s u f f i c i e n t to indicate that i s o l a t e d populations of marine sticklebacks can be found i n freshwater, and i f b a r r i e r s l a s t long enough fo r i s o l a t i n g mechanisms to develop, reproductive i s o l a t i o n w i l l come about, and species formed. Provided other plausible alternatives can be reasonable excluded, geographic speciation may be accepted as most l i k e l y . However, there are other alternatives that may apply to sticklebacks. Sympatric speciation r e s u l t i n g from disruptive s e l e c t i o n i s possible, and furthermore geographic speciation i s not without d i f f i c u l t i e s . Sticklebacks have a very widespread but interrupted 107 d i s t r i b u t i o n i n b o r e a l r e g i o n s . The d i s t r i b u t i o n o f t r a c h u r u s i s a l m o s t c o n t i n u o u s around the hemisphere b e i n g i n t e r r u p t e d o n l y i n n o r t h e a s t e r n R u s s i a (Berg, 1965) . L e i u r u s , however, has a v e r y d i s r u p t e d d i s t r i b u t i o n o c c u r r i n g i n Europe , G r e e n l a n d , I c e l a n d , B a f f i n I s l a n d , the A t l a n t i c C o a s t o f N o r t h A m e r i c a , the P a c i f i c C o a s t o f N o r t h A m e r i c a , Queen C h a r l o t t e I s l a n d , K o d i a k , the A l e u t i a n s i n c l u d i n g remote i s l a n d s l i k e A g a t t u , Shemya, S t . Matthew, P r i b o l o f s , B e r i n g I s l a n d , A t t u , and f i n a l l y J a p a n . (Evermann and G o l d s b o r o u g h , 1907; B e r g , 1965; M u n z i n g , 1963; d a t a f rom the N a t i o n a l Museum o f Canada and the I n s t i t u t e o f F i s h e r i e s , U . B . C . ) . I f s p e c i a t i o n were g e o g r a p h i c and the f r e s h w a t e r race a rose i n i s o l a t i o n a t a g i v e n p l a c e , how c o u l d i t t h e n expand i t s d i s t r i b u t i o n i n the f r e s h w a t e r s t h r o u g h o u t so v a s t an a r e a on s e v e r a l c o n t i n e n t s and on many remote i s l a n d s ? L e i u r u s i s adapted to f r e s h w a t e r and i s unknown from the s e a . T o l e r a n c e f o r s a l t water i s not g r e a t (Heuts, 1947), so t h a t i t i s i m p l a u s i b l e t o - s u g g e s t t h a t i t a rose on one c o n t i n e n t and s p r e a d to o t h e r s and to many remote i s l a n d s . However s p e c i a t i o n o c c u r r e d i t seems i n e s c a p a b l e t h a t t r a c h u r u s , w h i c h i s known from the open ocean (Bigelow and S c h r o e d e r , 1953), gave r i s e to l e i u r u s more than once , p r o b a b l y many t i m e s . I t i s d i f f i c u l t to b e l e i v e t h a t g e o g r a p h i c s p e c i a t i o n ( d i v e r g e n c e i f you l i k e ) c o u l d o c c u r many t imes such t h a t t r a c h u r u s produces many l e i u r u s p o p u l a t i o n s . M a y r ' s (1963) remarks c o n c e r n i n g the p r o b a b i l i t y t h a t s u c c e s s f u l g e o g r a p h i c s p e c i a t i o n w i l l o c c u r once are as f o l l o w s , "Most s p e c i e s bud 108 o f f peripheral i s o l a t e s at regular i n t e r v a l s . Nearly a l l of them either r e e s t a b l i s h contact with the parental species or else die out." And then, "Speciation i s a r i s k y process. The improver-ishment of the gene pool and the genetic i n s t a b i l i t y that accompanies i t are far more l i k e l y to lead to disaster than to success." However, Mayr shows there i s ample time i n the h i s t o r y of the earth to account for a l l species as a r e s u l t of geographic speciation; the point at issue i s that i n any given species the chances that a geographic i s o l a t e would successfully speciate are very small. If, as Mayr contends, th i s i s so i t seems even more implausible that trachurus gave r i s e to leiurus many times. Furthermore, i t seems u n l i k e l y that permanent b a r r i e r s necessary for geographic speciation would be at a l l common on extremely small islands l i k e Attu and others. Indeed, there are very few streams present on such islands that trachurus could use to breed i n , and thus become i s o l a t e d . Even i f they did become is o l a t e d b a r r i e r s on such islands would probably be very temporary. As mentioned, sympatric speciation by disruptive s e l e c t i o n i s a possible a l t e r n a t i v e . Mather (1955) discussed the expected consequences of disruptive s e l e c t i o n and pointed out that i t could e i t h e r e s t a b l i s h a polymorphism i n a population or i t could promote genetic i s o l a t i o n . When two optimal phenotypes are favoured i n a population and each i s dependent on the other, as i n sex or Batesian mimicry, polymorphism w i l l r e s u l t . If the two favoured optima are independent of one another divergence and i s o l a t i o n w i l l r e s u l t . 109 Obviously disruptive s e l e c t i o n has extremely important implications i n evolution and Thoday i n a long series of experiments has v e r i f i e d Mather's predictions (see Thoday, 1963). Thoday, using Drosophila melanogaster demonstrated that disruptive s e l e c t i o n has the following e f f e c t s : 1. promotes genetic v a r i a b i l i t y 2. can produce polymorphism 3. produces genetic divergence and i s o l a t i o n 4. produces coupling linkages His research has also shown that s t a b i l i z i n g s e l ection produces repulsion linkages. Furthermore, Clarke and Sheppard (1962) have presented strong evidence that disruptive s e l e c t i o n operates i n natural populations to perfect mimicry i n the polymorphic females of Papilio dardanus by perfecting dominance of the switching genes that control the polymorphism. With respect to sympatric speciation, 2) l i s t e d above i s not important and w i l l not be considered, but the remaining facts are important when sympatric speciation i n sticklebacks i s discussed. Mather (1955) has made i t clear that sympatric speciation as a r e s u l t of disruptive s e l e c t i o n depends on three c r i t e r i a : 1. The habitat to which each optimum phenotype i s adapted must p e r s i s t . 2. Each optimum phenotype must be independent of the other. 3. The phenotypes i n each habitat must be s u f f i c i e n t l y d i s t i n c t that d i f f e r e n t s e l e c t i v e forces can operate 110 on each. A l l the c r i t e r i a are s a t i s f i e d i n sticklebacks. The habitats to which trachurus and leiurus are adapted (sea and freshwater) do p e r s i s t i n time, independent phenotypes may arise from trachurus as a r e s u l t of recombination and crossing-over, and the phenotypes i n each habitat may be s u f f i c i e n t l y d i s t i n c t as a res u l t , again, of recombination and crossing-over i n trachurus populations. In Thoday's experiments disruptive s e l e c t i o n was applied to both ends of the normal curve for sternopleural chetae (a polygenic character). That i s , he selected for individu a l s with high chetae number and low chetae number. These highs and lows were put into v i a l s together and l e f t to mate eithe r assortatively, d i s a s s o r t a t i v e l y , or randomly. Within 12 to 16 generations divergence between highs and lows had occurred and reproductive i s o l a t i o n , as a r e s u l t of mate preference and probably hybrid i n f e r i o r i t y , was well developed. It i s important to r e a l i z e that the aspect, or aspects, of the phenotype that disruptive s e l e c t i o n operates on i s i r r e v e l a n t . I t i s only important that the phenotypic character have a polygenic basis, such as plates, g i l l rakers, and chetae. It i s obvious that trachurus populations carry large stores of concealed genetic v a r i a b i l i t y as a l l populations of outbreeding animals do (Dobzhansky, 1955; Dempster, 1955; Mayr, 1963). This i s e s p e c i a l l y obvious i n trachurus because of much geographic v a r i a t i o n , and regardless of how i t gave r i s e to leiurus, i f speciation occurred many times this demonstrates the large stores of v a r i a b i l i t y trachurus must carry. I l l Polygenic v a r i a t i o n i s most l i k e l y stored as balanced polygenic systems within the chromosomes (Mather and Harrison, 1949; Mather, 1956; Thoday, 1963). This v a r i a t i o n w i l l mostly be stored i n the heterozygous state and balanced. This pot e n t i a l genetic v a r i a b i l i t y stored i n balanced polygenic systems gives genetic f l e x i b i l i t y and the a b i l i t y to adapt to change i n environment because i t can be released slowly over many generations as a r e s u l t of crossing-over and recombination between linked polygenes (Mather, 1943). Sympatric speciation i n sticklebacks could occur i n the following way. Trachurus spends most of i t s l i f e i n the sea but moves into freshwater to breed. Genetic v a r i a t i o n stored i n balanced polygenic systems may occasionally, and over long periods, give r i s e to individuals better adapted to freshwater as a r e s u l t of crossing-over and recombination. Such individuals may remain i n freshwater for longer periods because they are better adapted. At t h i s point two optimal phenotypes are present; natural s e l e c t i o n w i l l continue to increase adaptation to freshwater, and disruptive s e l e c t i o n w i l l promote divergence and i s o l a t i o n despite sympatry (Thoday, 1963). A .specific example makes thi s clear and g i l l rakers (a polygenic character) may be used. In trachurus crossing-over and recombination within or between balanced polygenes that control raker number may occasionally occur, producing individuals with fewer rakers that are better adapted to feeding i n freshwater. These indivi d u a l s could then remain i n freshwater (as trachurus sometimes do i n winter). These o r i g i n a l recombinants may 112 occasionally interbreed with trachurus but lower raker numbers would s t i l l be preserved because disruptive s e l e c t i o n promotes coupling linkages, holding the genotypes c o n t r o l l i n g lower raker numbers together once they are formed (Thoday, 1963). The intermediates that may be formed from interbreeding would be selected against because they are less well adapted than trachurus i n the sea and less well adapted than individuals with lower raker number i n freshwater. Since individuals with lower raker numbers w i l l be preserved (coupling linkages), s e l e c t i o n w i l l continue to perfect t h e i r adaptation, and s e l e c t i o n w i l l be intense i n the divergent but stable freshwater environment. Selection and adaptation w i l l continue to produce individuals with lower raker numbers that are s t i l l better adapted, and w i l l continue to eliminate less well adapted intermediates i f they occur. But interbreeding w i l l be progressively less because a l l the while disruptive s e l e c t i o n i s operating. Where the two races (or divergent populations) are sympatric, disruptive s e l e c t i o n w i l l favour two optimal phenotypes (lows and highs) and intermediates w i l l be selected against (Thoday, 1963). The newly arisen race w i l l eventually become so well adapted to the new habitat that ec o l o g i c a l i s o l a t i o n between races w i l l r e s u l t . In t h i s example a single polygenic characters, g i l l rakers, i s used. In f a c t s e l e c t i o n operates on the t o t a l phenotype (Mayr, 1963). If other polygenic characters that are adaptive i n freshwater are considered (body shape, behavioural t r a i t s , p hysiological properties) and disruptive s e l e c t i o n operates on a l l , preserving these characters (coupling linkages) and s e l e c t i n g 113 against less well adapted intermediates i n any or a l l characters, then divergence and i s o l a t i o n w i l l ensue. In fact, t h i s process of disruptive s e l e c t i o n helps to explain the d i f f i c u l t problem of why swamping does not occur i n hybrid zones of sticklebacks. Disruptive s e l e c t i o n would prevent swamping because i t promotes divergence and i s o l a t i o n , not swamping. Moreover, i f disruptive s e l e c t i o n were operating i n hybrid zones i t s e f f e c t s would be most intense i n or near the hybrid zone where interbreeding occurs. And i t would produce divergence i n polygenic characters, l i k e plates. This increased divergence i s i n f a c t found, and again, disruptive s e l e c t i o n explains a d i f f i c u l t problem. This d i f f i c u l t problem concerns the f a c t that leiurus nearest the hybrid zone have lower plate counts than normal, and trachurus i n the hybrid zone have higher plate counts than normal. This i s the reverse to be expected i f introgression were occurring. It i s expected i f disruptive s e l e c t i o n i s occurring. Disruptive s e l e c t i o n could promote the divergence i n t h i s polygenic character, and i t would preserve the divergence (coupling linkages). It i s i n t e r e s t i n g to note that i n another case of reported sympatric speciation (Ford, 1964) a reverse c l i n e was found with distance from the hybrid zone. Also t h i s case was concerned with two populations (Maniola jurtina) meeting across two habitats. In summary, sympatric speciation by disruptive s e l e c t i o n cannot be excluded as a plausible explanation of sticklebacks speciation. I t i s consistent with the widespread 114 but disjunct d i s t r i b u t i o n of leiurus, and e s p e c i a l l y so i f that race has arisen from trachurus more than once, because th i s mode of speciation does not contain the same element of r i s k that geographic speciation does. There i s no homozygosity and impoverishment of the gene pool that makes geographic speciation so r i s k y (Mayr, 1963). On the contrary, disruptive s e l e c t i o n promotes genetic f l e x i b i l i t y (Thoday, 1963) providing for raw material on which natural s e l e c t i o n can operate i n a new and d r a s t i c a l l y d i f f e r e n t environment. Disruptive s e l e c t i o n seems therefore a more plausible explanation for repeated development of freshwater forms from a marine ancestor, without invoking a remarkable series of coincidences involving temporary geographic b a r r i e r s i n each i s o l a t e d stream now occupied by both types. TAXONOMIC STATUS OF.LEIURUS AND TRACHURUS Mayr (1963) defines species as "Groups of a c t u a l l y or p o t e n t i a l l y interbreeding populations which are reproductively i s o l a t e d from other such groups." Sibley (1961) c l a r i f i e d the d e f i n i t i o n with respect to instances where hyb r i d i z a t i o n occurs, pointing out that one must attempt to determine whether or not hybrids are at a selec t i v e disadvantage r e l a t i v e to parental types. If hybrids are at a s e l e c t i v e disadvantage the s p e c i f i c status of the hybridi z i n g populations i s insured - that i s , gene flow w i l l be more or less completely blocked between populations by natural s e l e c t i o n . 115 Leiurus and trachurus populations studied i n the L i t t l e Campbell River and on Vancouver Island f u l f i l l the species d e f i n i t i o n . There i s considerable genetic divergence between the two, i s o l a t i n g mechanisms are well developed, and they are reproductively i s o l a t e d . There i s considerable morphological divergence between the species, and most characters investigated have a polygenic b a s i s . Other d i s t i n c t i o n s such as ecological i s o l a t i o n , seasonal i s o l a t i o n , physiological and behavioural attributes l i k e anadromy, a l l exemplify considerable genetic divergence. Iso l a t i n g mechanisms have already been discussed. I t remains only to emphasize the e f f i c i e n c y with which natural s e l e c t i o n blocks gene flow between the species. In the L i t t l e Campbell River where hyb r i d i z a t i o n i s extensive, gene glow i s blocked withone one mile, and each species remains d i s t i n c t on either side of t h i s b r i e f zone. There i s no i n d i c a t i o n that introgression occurs, i n f a c t the reversed c l i n e i n leiurus demonstrates that stringent s e l e c t i o n counters genetic exchange. In Bonsall Creek gene flow i s prevented within 200 feet, also . evidence of very stringent s e l e c t i o n . Here too, where contiguous populations occur over an interface between habitats, reproductive i s o l a t i o n of each species i s preserved. Farther a f i e l d , an examination of specimens from Alaska to Oregon ( i n s t i t u t e of Fisheries, U.B.C.) reveals that the morphological c h a r a c t e r i s t i c s that distinguish the two species i n the L i t t l e Campbell River and Vancouver Island also di s t i n g u i s h other populations. An examination of the extensive 116 l i t e r a t u r e provides confirmation, and further indicates that the b i o l o g i c a l attributes of the two remain d i s t i n c t from Europe (Heuts, 1947; Munzing, 1963), and Russia (Berg, 1965), to the P a c i f i c . C l e a r l y although leiurus and trachurus are sympatric over a vast sphere they remain d i s t i n c t and reproductively isolated; they are "good" species. However, the taxonomy may be resolved, sticklebacks furnish e x c i t i n g material for evolutionary studies. Evidence i s accumulating that there are s i b l i n g species within the complex. Gasterosteus wheatlandi on the A t l a n t i c Coast i s one such species (Hubbs, 1929; Scott and Crossman, 1964), and another possible species has been uncovered by J. D. McPhail (personal communication) on the P a c i f i c Coast. A possible t h i r d species on the Queen Charlotte Islands i s currently being investigated by E. E. Moodie and myself. The p r o b a b i l i t y of an independent o r i g i n of leiurus from trachurus many times gives one cause to pause. The morphology of leiurus i s quite uniform throughout i t s range, reproductive i s o l a t i o n i s maintained, and so one wonders i f the i s o l a t i n g mechanisms that separate leiurus and trachurus are everywhere the same. Are the freshwater forms reproductively i s o l a t e d from one another? In a word, has sel e c t i o n independently and on many occasions produced as uniform a genotype as i t has a phenotype? Sticklebacks o f f e r wide scope for a better understanding of speciation and the mechanisms of evolution. 117 CONCLUSIONS Hybridization i s extensive between the marine stickleback (trachurus)and the freshwater form (leiurus) but i s r e s t r i c t e d to very narrow hybrid zones i n the L i t t l e Campbell River and on Vancouver Island. A morphological analysis provides firm circumstantial evidence that hybrids are p l e n t i f u l and that backcrossing occurs. This i s confirmed by uniform rearing experiments i n a l l combinations. Most backcrossing i s to the freshwater race. An investigation of i s o l a t i n g mechanisms as outlined by Mayr reveals that neither behavioural nor genetic b a r r i e r s are e f f e c t i v e blocks to hybridization, and thus there i s no means to prevent h y b r i d i z a t i o n where they come together. But ecol o g i c a l i s o l a t i n g mechanisms provide a very powerful b a r r i e r that greatly reduces opportunity to hybridize. This i s enforced to same extent by p a r t i a l seasonal i s o l a t i o n , but ear l y breeding migrants make a major contribution to interbreeding i n the L i t t l e Campbell River. Several factors probably operate to cause very narrow hybrid zones i n sticklebacks, including a sedentary habit, habitat preference, and hybrid i n f e r i o r i t y outside the hybrid zone (although none could be detected inside the zone). The complex of i s o l a t i n g mechanisms found could well produce stable hybrid zones for long periods, and i s discussed. Reinforcement of eco l o g i c a l i s o l a t i n g mechanisms probably occurs, and Moore's c r i t i c i s m of the spread of reinforced genotypes outside the hybrid zone would not apply 118 i n t h i s instance. Reinforcement of e c o l o g i c a l i s o l a t i o n would r e s u l t i n exclusion between races, not b i o t i c sympatry. Adaptation and consequent ecological i s o l a t i n g mechanisms are the cause of hybrid i n f e r i o r i t y . In t h i s instance pre and postmating i s o l a t i n g mechanisms cannot be separated, for they go hand i n hand. This i s discussed i n r e a l t i o n to Mayr's c l a s s i f i c a t i o n of i s o l a t i n g mechanisms. Geographic speciation i s possible but problematical; sympatric speciation cannot be excluded as a plausible explanation and could have occurred as a r e s u l t of disruptive s e l e c t i o n . Furthermore, d i s t r i b u t i o n a l patterns of leiurus can be explained most reasonably as a r e s u l t of several, perhaps many, independent ori g i n s from trachurus. The two forms f u l f i l l the species d e f i n i t i o n of Mayr and remain reproductively isolat e d , have well developed i s o l a t i n g mechanisms, and show considerable genetic divergence, much of i t polygenic. 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