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Maintenance of reproductive isolation between hybridizing populations of the peamouth chub, Mylocheilus… Aspinwall, Nevin 1968

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THE MAINTENANCE  OF R E P R O D U C T I V E ISOLATION B E T W E E N  H Y B R I D I Z I N G P O P U L A T I O N S OF T H E P E A M O U T H CHUB, M Y L O C H E I L U S CAURPNUM AND T H E REDSIDE SHINER, RICHARDSONIUS B A L T E A T U S  by NEVIN ASPINWALL B . S c , U n i v e r s i t y of Washington, 1962 M . S c , U n i v e r s i t y of Washington, 1965  'A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L M E N T O F THE REQUIREMENTS  FOR THE DEGREE  OF  D O C T O R OF P H I L O S O P H Y  i n the D e p a r t m e n t of Zoology  We a c c e p t this 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 standard  T H E U N I V E R S I T Y OF BRITISH September, 1968  COLUMBIA  In p r e s e n t i n g an the  thesis  advanced degree at Library  I further for  this  shall  the  in p a r t i a l  f u l f i l m e n t of  University  of  make i t f r e e l y  agree that  permission  s c h o l a r l y p u r p o s e s may  by  his  of  this  written  representatives. thesis  be  available  for  for extensive  granted  by  the  It i s understood  for financial  gain  permission.  Department The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  British  Columbia  shall  requirements  Columbia,  Head o f my  be  I agree  r e f e r e n c e and copying of  that  not  the  that  Study.  this  thesis  Department  c o p y i n g or  for  or  publication  allowed without  my  ii  ABSTRACT The m o d e r n species concept ( M a y r , 1963 ) s t r e s s e s the i n t e r b r e e d i n g w i t h i n , and the r e p r o d u c t i v e i s o l a t i o n between, species. breeding"  However, " i n t e r -  i s not s t r i c t l y an i n t r a s p e c i f i c c h a r a c t e r i s t i c since n u m e r o u s  i n t e r s p e c i f i c h y b r i d s have been r e p o r t e d , e s p e c i a l l y among the temperate freshwater fishes.  In this i n v e s t i g a t i o n , h y b r i d i z i n g ( i n t e r b r e e d i n g ) popu-  l a t i o n s of the peamouth chub, M y l o c h e i l u s c a u r i n u m ( R i c h a r d s o n ) , and the r e d s i d e s h i n e r , R i c h a r d s o n i u s balteatus ( R i c h a r d s o n ) f r o m Stave L a k e , B r i t i s h C o l u m b i a , w e r e studied to determine: (1) i f i n t e r b r e e d i n g between them was r e s u l t i n g i n the s w a m p i n g (lack of r e p r o d u c t i v e i s o l a t i o n ) of t h e i r gene pools, and (2) i f swamping was absent, what i s o l a t i n g m e c h a n i s m s were operative? To detect the p r e s e n c e o r absence of swamping, two approaches w e r e used.  The f i r s t m e a s u r e d shifts i n m e a n s of c e r t a i n m o r p h o l o g i c a l c h a r a c t e r s  f o r the two s p e c i e s w i t h i n and outside the a r e a of h y b r i d i z a t i o n . d e t e r m i n e d the frequency of v a r i o u s h y b r i d generations.  The second  If swamping i s  not o c c u r r i n g , the frequency of h y b r i d b a c k c r o s s i n d i v i d u a l s should d e c r e a s e as b a c k c r o s s i n g continues..  B o t h a p p r o a c h e s i n d i c a t e d that swamping i s  absent between M y l o c h e i l u s and R i c h a r d s o n i u s . i In the absence of swamping, i s o l a t i n g m e c h a n i s m s between the two s p e c i e s w e r e examined.  Seasonal, t e m p o r a l (diel), s p a t i a l , and e t h o l o g i -  c a l p r e m a t i n g i s o l a t i n g m e c h a n i s m s do not appear to be effective since M y l o c h e i l u s , R i c h a r d s o n i u s , and t h e i r h y b r i d s spawn at the same time and place w i t h i n D e v i l s Creek," a m a j o r spawning a r e a i n Stave Lake.  iii E g g and f r y s u r v i v a l of c r o s s e s i n v o l v i n g h y b r i d i n d i v i d u a l s w e r e m e a s u r e d under e x p e r i m e n t a l conditions to d e t e r m i n e if they serve as post-mating i s o l a t i n g m e c h a n i s m s .  F^ h y b r i d m a l e s a r e p a r t i a l l y s t e r i l e  as d e m o n s t r a t e d by the poor egg s u r v i v a l of c r o s se s i n v o l v i n g them.  How-  ever, egg s u r v i v a l of h y b r i d f e m a l e s when b a c k c r o s s e d with M y l o c h e i l u s and R i c h a r d s o n i u s  m a l e s was c o m p a r a b l e to the p a r e n t a l s p e c i e s ' c r o s s e s .  Thus, f e r t i l i t y i s not c o n s i d e r e d an e f f e c t i v e i s o l a t i n g m e c h a n i s m .  The  e x p e r i m e n t s t e s t i n g the f r y s u r v i v a l of the p a r e n t a l s p e c i e s , r e c i p r o c a l F j h y b r i d s , and h y b r i d b a c k c r o s s e s to M y l o c h e i l u s ( = B CM) y i e l d a clue to what i s o l a t i n g m e c h a n i s m prevents swamping.  The f r y s u r v i v a l of  r e c i p r o c a l F^ h y b r i d f r y do not d i f f e r s i g n i f i c a n t l y f r o m M y l o c h e i l u s fry.  In c o n t r a s t , the s u r v i v a l of h y b r i d b a c k c r o s s to M y l o c h e i l u s f r y  is approximately  20 % l e s s than e i t h e r r e c i p r o c a l F j h y b r i d s o r M y l o c h e i l u s  f r y a f t e r only 48 days r e a r i n g .  A d d i t i o n a l l y , 11. 5 % of the s u r v i v i n g B C M  fry possess gross abnormalities.  Thus, h y b r i d i n f e r i o r i t y i s d e m o n s t r a t e d  experimentally. C i r c u m s t a n t i a l evidence was a l s o gathered f r o m Stave L a k e i n 1967 w h i c h suggests the i n f e r i o r i t y of B C M  fry.  R e l a t i v e to M y l o c h e i l u s ,  B C M s w e r e five t i m e s m o r e abundant as f i n g e r l i n g s than as adults. L i t t l e i n f o r m a t i o n was gathered on h y b r i d b a c k c r o s s e s to R i c h a r d s o n i u s . They, a p p e a r e d s c a r c e as f i n g e r l i n g s as w e l l as adults.  iv  T A B L E OF  CONTENTS PAGE  ABSTRACT  i i  T A B L E OF CONTENTS  iv  LIST O F T A B L E S  vii  LIST O F F I G U R E S  ix  ACKNOWLEDGEMENTS  xi  INTRODUCTION  1  G E N E R A L M A T E R I A L S AND METHODS  7  D e s c r i p t i o n of Study A r e a  7  Sampling  9  REPRODUCTIVE ISOLATION B E T W E E N M YLOCHEILUS AND RICHARDSONIUS  10  Morphological Approach  10  Morphological-Biochemical Approach  11  Search for B i o c h e m i c a l Characters  13  Electrophoretic Analysis  13  R e a r i n g of E x p e r i m e n t a l A n i m a l s M y l o c h e i l u s and R i c h a r d s o n i u s Outside the H y b r i d Zone  15 15  Muscle Proteins  15  M a l i c Dehydrogenase  18  Inheritance of B i o c h e m i c a l C h a r a c t e r s  18  Muscle Proteins  18  M a l i c Dehydrogenase (MDH)  25  Linkage  28  V  PAGE Inheritance of A n a l F i n R a y s ; S e p a r a t i o n of 2nd and Later Generation H y b r i d Backcrosses  30  L i n k a g e of B i o c h e m i c a l and M o r p h o l o g i c a l C h a r a c t e r s  33  S e p a r a t i o n of F\ H y b r i d s and 1st G e n e r a t i o n H y b r i d Backcrosses  36  Results  40  P r e d o r s a l / P r e p e l v i c Length R a t i o s (D/P) and L a t e r a l L i n e S c a l e s of M y l o c h e i l u s and R i c h a r d s o n i u s f r o m Stave L a k e and f r o m A r e a s Outside the H y b r i d Zone  40  Composition'of the H y b r i d P o p u l a t i o n i n Stave L a k e  40  ISOLATING MECHANISMS  47  M a t e r i a l s and M e t h o d s  47  Observations  47  Survival Experiments  49  E g g S u r v i v a l to H a t c h i n g  49  S u r v i v a l of Y o l k - s a c F r y to F i n g e r l i n g s  50  Results  51  Premating Isolating M e c h a n i s m s  51  Seasonal I s o l a t i o n  51  S p a t i a l and T e m p o r a l ( D i e l ) I s o l a t i o n  55  Ethological Isolation  59  Postmating Isolating Mechanisms  61  E g g S u r v i v a l to H a t c h i n g  61  S u r v i v a l of Y o l k - s a c F r y to F i n g e r l i n g s  65  A b n o r m a l i t i e s of E x p e r i m e n t a l A n i m a l s  67  1  vi  PAGE Sex R a t i o s of H y b r i d and P a r e n t a l Species' Populations  69  C o m p a r i s o n of F i n g e r l i n g and A d u l t H y b r i d A n a l F i n Ray Distributions  71  DISCUSSION  74  I n t r o g r e s s i o n , Swamping, and R e p r o d u c t i v e I s o l a t i o n  74  Isolating M e c h a n i s m s  78  Cause(s) of H y b r i d i z a t i o n and L a c k of R e i n f o r c e m e n t  86  S U M M A R Y AND CONCLUSIONS  91  L I T E R A T U R E CITED  95  vii LIST OF T A B L E S Table I.  IL  III.  IV.  V.  VI.  VII.  VIII.  IX.  X.  XL  XII.  XIII.  Page M u s c l e p r o t e i n and m a l i c dehydrogenase p a t t e r n s of M y l o c h e i l u s and R i c h a r d s o n i u s outside the h y b r i d zone (number of individuals).  17  M u s c l e p r o t e i n patterns of parents and progeny of c r o s s e s made i n 196.6 and 1967.  20  M a l i c dehydrogenase patterns of parents and progeny of c r o s s e s made i n 1967.  26,  C o r r e l a t i o n of m a l i c dehydrogenase and m u s c l e p r o tein p a t t e r n s of h y b r i d b a c k c r o s s e s to M y l o c h e i l u s .  29  The p r o p o r t i o n of v a r i o u s m u s c l e p r o t e i n patterns of p r e s u m e d F^ h y b r i d b a c k c r o s s e s to M y l o c h e i l u s i n r e l a t i o n to a n a l f i n r a y number.  34  The p r o p o r t i o n of v a r i o u s m a l i c dehydrogenase patterns of p r e s u m e d Fj^ h y b r i d b a c k c r o s s e s to M y l o c h e i l u s i n r e l a t i o n to a n a l f i n r a y number.  35  C l a s s i f i c a t i o n of h y b r i d types b a s e d on the c o m b i n a t i o n of m u s c l e p r o t e i n and m a l i c dehydrogenase patterns.  38  C o m p a r i s o n of m o r p h o l o g i c a l c h a r a c t e r s of M y l o c h e i l u s and R i c h a r d s o n i u s f r o m w i t h i n and outside the h y b r i d zone.  43  M u s c l e p r o t e i n and m a l i c dehydrogenase patterns of those i n d i v i d u a l s of the M y l o c h e i l u s - R i c h a r d s o n i u s gene pools w i t h <_ 8 o r > 15 a n a l f i n rays.  44  C o m p o s i t i o n of the M y l o c h e i l u s - R i c h a r d s o n i u s gene c o m p l e x of Stave L a k e i n 1967.  45  G i l l net catches of M y l o c h e i l u s , R i c h a r d s o n i u s , and t h e i r h y b r i d s f r o m D e v i l s C r e e k d u r i n g the spawning season of 1966.  53  G i l l net catches of M y l o c h e i l u s , R i c h a r d s o n i u s , and t h e i r h y b r i d s f r o m D e v i l s C r e e k d u r i n g the spawning season of 1967.  54  C o m p o s i t i o n of a s a m p l e of the spawning population f r o m D e v i l s C r e e k at 2245 hours(P.D.T.), June 16, 1966.  58  Vlll  Table XIV.  XV.  Page M e a n egg s u r v i v a l of e x p e r i m e n t a l c r o s s e s to hatching and tests of s i g n i f i c a n c e between o r d e r e d p a i r s of m e a n s using the Newman-Keuls p r o c e d u r e (Winer, 1962).  62  G r o s s a b n o r m a l i t i e s of f i s h u s e d i n the viabilitye x p e r i m e n t s f r o m the y o l k - s a c f r y to the f i n g e r l i n g stage.  68  ix LIST OF  FIGURES  The peamouth chub, M y l o c h e i l u s c a u r i n u m (above), r e d s i d e s h i n e r , R i c h a r d s o n i u s balteatus (below), and t h e i r h y b r i d (middle). A n a l f i n r a y d i s t r i b u t i o n of the M y l o c h e i l u s R i c h a r d s o n i u s gene pool f r o m Alouette and Stave L a k e s (1967 adults). Stave L a k e , B r i t i s h C o l u m b i a , and adjacent  waters.  S t a r c h - g e l e l e c t r o p h o r e s i s of m u s c l e p r o t e i n s f r o m M y l o c h e i l u s c a u r i n u m , R i c h a r d s o n i u s balteatus, and t h e i r h y b r i d s . S t a r c h - g e l e l e c t r o p h e r o g r a m s of m a l i c dehydrogenase f r o m M y l o c h e i l u s c a u r i n u m , R i c h a r d s o n i u s balteatus, and t h e i r h y b r i d s . A genetic m o d e l f o r the i n h e r i t a n c e of m u s c l e p r o t e i n s , zones 1 and 3, i n R i c h a r d s o n i u s and M y l o c h e i l u s , respectively, A n a l f i n r a y d i s t r i b u t i o n of 1966 and 1967 e x p e r i m e n t a l c r o s s e s combined. E l e c t r o p h e r o g r a m s of m u s c l e p r o t e i n and. m a l i c dehydrogenase patterns f r o m three n a t u r a l h y b r i d s f r o m Stave L a k e (1967 samples). The r a t i o of p r e d o r s a l to p r e p e l v i c length of M y l o c h e i l u s and R i c h a r d s o n i u s w i t h i n and outside the h y b r i d zone. L a t e r a l line scale d i s t r i b u t i o n of M y l o c h e i l u s and R i c h a r d s o n i u s w i t h i n and outside the h y b r i d zone. D i a g r a m of D e v i l s Creek, B r i t i s h Columbia. Seasonal d i s t r i b u t i o n of M y l o c h e i l u s , R i c h a r d s o n i u s and t h e i r h y b r i d s i n D e v i l s C r e e k f o r 1966 and 1967. R e l a t i o n s h i p between l o c a t i o n of spawning f i s h and the length of D e v i l s C r e e k f o r p a r t i c u l a r dates i n 1967.  X Figure 14.  15.  16.  17.  p  a  g  e  A n a l f i n r a y d i s t r i b u t i o n of f i n g e r l i n g s r e a r e d f r o m eggs c o l l e c t e d on June 20, 1966, f r o m an 18" square s e c t i o n of the "20 m e t e r " r i f f l e of D e v i l s Creek.-  60  S u r v i v a l of y o l k - s a c f r y to the f i n g e r l i n g stage under e x p e r i m e n t a l conditions.  66  T y p i c a l a b n o r m a l i t i e s of progeny f r o m h y b r i d b a c k c r o s s e s to M y l o c h e i l u s .  70  D i s t r i b u t i o n of-anal f i n r a y s f r o m "9" to "13" f o r 1967 f i n g e r l i n g s and f o r 1966 and 1967 adults.  73  xi ACKNOWLEDGEMENTS I w i s h to thank Dr. J. D. M c P h a i l , my  research supervisor, for  m a n y useful suggestions he made on the f i e l d r e s e a r c h and i n the p r e p a r a t i o n of the m a n u s c r i p t .  He was a l s o m o r e than generous i n f i n a n c i n g  this study, of n e c e s s i t y a c o s t l y one. Dr. C. C. L i n d s e y who  s e r v e d as my  T h a n k s a r e a l s o extended to s u p e r v i s o r f o r the f i r s t year.  Guidance was a l s o obtained f r o m other m e m b e r s of my  advising  committee, n a m e l y D r s . I. E. E f f o r d , H. D. F i s h e r , J. E. P h i l l i p s , N. J. W i l i m o v s k y .  The l a t t e r two m e m b e r s c r i t i c a l l y r e v i e w e d the  m a n u s c r i p t and suggested i m p o r t a n t changes. of my  committee,  and  I extend my  To a l l of the m e m b e r s  appreciation.  M a n y students of the Institute of F i s h e r i e s , U n i v e r s i t y of B r i t i s h Columbia, helped i n the f i e l d phase of the study.  I would e s p e c i a l l y like  to thank Dr. K. Stewart, now of the U n i v e r s i t y of M a n i t o b a , f o r the m a n y nights he a c c o m p a n i e d me  i n s t a l k i n g the spawning c y p r i n i d s of Stave  Lake. Mr.  L e n K e a r s l e y of Haney, B. C. , p e r m i t t e d the use of h i s house-  boat f o r l i v i n g q u a r t e r s and f o r a p o r t a b l e l a b o r a t o r y . My  wife, Joy, a s s i s t e d me  I thank him.  w i t h m u c h of the f i e l d work.  Additionally,  she edited the m a n u s c r i p t and p r e p a r e d m o s t of the f i g u r e s of the thesis. To h e r , I e x p r e s s deep gratitude. F i n a l l y , thanks a r e given to my nights d u r i n g the f i e l d season.  son, E r i c , who kept me  awake m a n y  1  INTRODUCTION The m o d e r n concept of " s p e c i e s " e n v i s i o n s them to be". . . groups of a c t u a l l y o r p o t e n t i a l l y i n t e r b r e e d i n g populations w h i c h a r e r e p r o d u c tive ly i s o l a t e d f r o m other such groups. " ( M a y r , 1963).  T h i s concept  s t r e s s e s the i n t e r b r e e d i n g w i t h i n , and the r e p r o d u c t i v e i s o l a t i o n between, species.  W i t h i n the l a s t t h i r t y y e a r s , Dr. C a r l . L. Hubbs and h i s c o l l e a g u e s  d e m o n s t r a t e d that i n t e r b r e e d i n g between  supposedly v a l i d s p e c i e s i s c o m m o n  among f r e s h w a t e r f i s h e s , and s c o r e s of h y b r i d s a r e r e p o r t e d among the Catostomidae, C e n t r a r c h i d a e , and C y p r i n i d a e . supposedly valid,  Is i n t e r b r e e d i n g between  species r e c o n c i l a b l e w i t h the m o d e r n s p e c i e s concept  (Dobzhansky, 1951; Sibley, 1961; Bigelow,  1965; and Hagen, 1967)?  question has i n t e r e s t e d e v o l u t i o n i s t s f o r m a n y y e a r s and,  This  consequently,  they have made c o n c e r t e d e f f o r t s to understand the r e l a t i o n s h i p between h y b r i d s , h y b r i d zones, and r e p r o d u c t i v e i s o l a t i o n . ^ T o c l a r i f y this r e l a t i o n ship, m e c h a n i s m s w h i c h m a i n t a i n the genetic i n t e g r i t y ( r e p r o d u c t i v e i s o l a t i o n ) •  between two s p e c i e s m u s t be understood.  M a y r (1963) d i v i d e s these into  p r e m a t i n g and postmating i s o l a t i n g m e c h a n i s m s , a d i v i s i o n based on the o r d e r i n w h i c h they operate. When two c l o s e l y r e l a t e d , g e o g r a p h i c a l l y i s o l a t e d , populations r e e s t a b l i s h contact, there a r e at l e a s t four p o s s i b l e outcomes:  1) the two  1)".. r e f e r s to the p r o t e c t i v e d e v i c e s of a h a r m o n i o u s l y coadapted gene pool against d e s t r u c t i o n by genotypes f r o m other gene pools. " ( M a y r , 1963).  ,2 populations do not  i n t e r b r e e d because of effective p r e m a t i n g i s o l a t i n g  m e c h a n i s m s , and so m a i n t a i n the genetic i n t e g r i t y of the populations; 2) the two populations do i n t e r b r e e d but s e l e c t i o n a g a i n s t the h y b r i d s m a i n t a i n s the genetic i n t e g r i t y of the populations; 3) the two populations i n t e r b r e e d and c e r t a i n r e c o m b i n a n t types a r e at a s e l e c t i v e advantage and, t h e r e f o r e , p r o v i d e new genes to the gene pool(s) of one o r both populations ( i n t r o g r e s s i o n ). In such c a s e s , r e p r o d u c t i v e i s o l a t i o n i s m a i n t a i n e d between the populations because the i n t r o g r e s s e d genes do not d e s t r o y — b u t i n c r e a s e --the h a r m o n y of the coadapted gene pools; o r 4) the populations i n t e r b r e e d , f u s i n g into a single population (swamping), and genetic i n t e g r i t y i s lost. A c c o r d i n g to the species d e f i n i t i o n of M a y r , the populations i n s i t u ations (1), (2), and (3) have attained s p e c i f i c status. However, the d i s t i n c t i o n between (2), s p e c i f i c status, and (4), swamping, i s d i f f i c u l t to m a k e u n l e s s the populations have been o b s e r v e d f o r some time, o r u n l e s s i n the f o r m e r case s e l e c t i o n against the h y b r i d s has been demonstrated. The peamouth chub, M y l o c h e i l u s c a u r i n u m ( R i c h a r d s o n ) , and the r e d side s h i n e r , R i c h a r d s o n i u s balteatus ( R i c h a r d s o n ) , a r e two c o m m o n c y p r i n i d f i s h e s i n B r i t i s h C o l u m b i a and the northwe s t e r n U n i t e d States, and the two s p e c i e s c o e x i s t i n m a n y l o c a l i t i e s without i n t e r b r e e d i n g ( F i g .  1).  In fact, it i s d i f f i c u l t to find l a r g e a r e a s w h e r e t h e i r d i s t r i b u t i o n s do not overlap.  Two such a r e a s a r e V a n c o u v e r Island and the  Sechelt P e n i n s u l a  2) I n t r o g r e s s i o n i s ". . . an i n f i l t r a t i o n of the g e r m p l a s m of one s p e c i e s into that of another. " ( A n d e r s o n and H'ubricht, 1938).  3  F i g u r e 1.  The p earn oath chub, M y l o c h e i l u s c a u r i n u m  (above),  r e d s i d e s h i n e r , R i c h a r d s o n i u s balteatus (below), and t h e i r h y b r i d (middle). A l l three f i s h are i n spawning c o l o r a t i o n .  4 w h e r e only M y l o c h e i l u s  i s found.  In one l o c a l i t y , Stave L a k e , B r i t i s h  C o l u m b i a , M y l o c h e i l u s and R i c h a r d s o n i u s ization.  engage i n extensive h y b r i d -  The h y b r i d i s a l s o depicted i n F i g u r e 1.  This hybridization is  c l e a r l y d e m o n s t r a t e d by a c o m p a r i s o n of the a n a l f i n r a y d i s t r i b u t i o n s (Fig. 2) of the c o m b i n e d M y l o c h e i l u s - R i c h a r d s o n i u s  gene pools between  Stave L a k e (where h y b r i d s o c c u r ) and adjacent Alouette L a k e (where h y b r i d s a r e absent).  T h e r e i s no o v e r l a p i n a n a l r a y n u m b e r between M y l o c h e i l u s  (7-8 a n a l r a y s ) and R i c h a r d s o n i u s  (14-22 a n a l r a y s ) i n Alouette L a k e , but  i n Stave L a k e although the a n a l f i n ray d i s t r i b u t i o n i s b i m o d a l , there a r e m a n y i n d i v i d u a l s w i t h an i n t e r m e d i a t e n u m b e r of a n a l r a y s (9-12).  These  i n d i v i d u a l s w i t h 9 to 12 a n a l f i n r a y s a r e h y b r i d s . I n i t i a l l y , it a p p e a r e d that not only w e r e the two s p e c i e s h y b r i d i z i n g i n Stave Lake, but a l s o that they w e r e swamping. supporting this v i e w i s :  C i r c u m s t a n t i a l evidence  1) the m a j o r i t y of spawning M y l o c h e i l u s  L a k e , the h y b r i d zone, a r e u n u s u a l l y s m a l l (« 120 mm) R i c h a r d s d n i u s i n size(«100 mm).  i n Stave  and r e s e m b l e m a t u r e  In c o n t r a s t , spawning M y l o c h e i l u s  from  other a r e a s , i n c l u d i n g the n e a r b y ( a n d at one time sympatric) . M y l o c h e i l u s f r o m l o w e r Stave R i v e r , a r e u s u a l l y m u c h l a r g e r («200 mm); Mylocheilus  2)  i n the h y b r i d zone begin spawning a p p r o x i m a t e l y June 1,  w h e r e a s populations outside the h y b r i d zone spawn m u c h e a r l i e r . f r o m the Alouette and Stave R i v e r s spawn i n late A p r i l sonius spawn f r o m late M y a  and L i n d s e y , 1959)-  Mylocheilus  or e a r l y May.  Richard -  to August i n B r i t i s h C o l u m b i a ( C a r l , Clemens,  Both o b s e r v a t i o n s indicate that i n the h y b r i d zone  s i g n i f i c a n t changes have o c c u r r e d i n the M y l o c h e i l u s  population that b r i n g  ALOUETTE  F i g u r e 2.  LAKE  A n a l f i n r a y d i s t r i b u t i o n of the M y l o c h e i l u s - R i c h a r d s o n i u s f r o m A l o u e t t e and S t a v e L a k e s ( 1 9 6 7 a d u l t s ) . 25 a n d 38 rain s t r e t c h m e s h g i l l n e t s .  gene pool  Samples were collected with  i t c l o s e r to R i c h a r d s o n i u s and suggest that swamping m i g h t be i n p r o g r e s s . The purpose of the p r e s e n t sutdy was  to d e t e r m i n e w h i c h of the three  h y b r i d situations d e s c r i b e d above best f i t s the Stave L a k e populations of M y l o c h e i l u s and R i c h a r d s o n i u s , and i f r e p r o d u c t i v e i s o l a t i o n e x i s t s , to attempt to d e t e r m i n e what i s o l a t i n g m e c h a n i s m ( s ) a r e o p e r a t i v e ?  7 G E N E R A L M A T E R I A L S AND METHODS D e s c r i p t i o n of Study A r e a Stave L a k e and one of i t s s m a l l t r i b u t a r y s t r e a m s , D e v i l s C r e e k w e r e the p r i m a r y study a r e a s ( F i g . 3). O r i g i n a l l y 19 k m i n length, the lake was  e n l a r g e d to 30 k m w i t h the c o m p l e t i o n of h y d r o e l e c t r i c f a c i l i t i e s i n 1924.  The lake now c o n s i s t s of an upper p o r t i o n , 19 k m i n length, and a l o w e r p o r t i o n of 11 k m that c o r r e s p o n d s to the flooded Stave R i v e r channel.  The  flooded portions contain l a r g e stands of s u b m e r g e d f o r e s t . A n n u a l f l u c t u ations i n the lake l e v e l of 9 m e t e r s a r e not u n c o m m o n and c e r t a i n t r i b u t a r y s t r e a m s a r e m a r k e d l y a l t e r e d by these fluctuations.  Stave L a k e i s a n  o l i g o t r o p h i c lake containing at l e a s t nine s p e c i e s of f i s h i n a d d i t i o n to the a f o r e m e n t i o n e d hybrids.  They are:  spine s t i c k l e b a c k , G a s t e r o s t e u s squawfish,  kokanee, Oncorhynchus n e r k a ,  three-  aculeatus, cutthroat trout, Salmo c l a r k i i ,  P t y c h o c h e i l u s oregonense, peamouth chub, M y l o c h e i l u s c a u r i n u m ,  redside s h i n e r , R i c h a r d s o n i u s balteatus, l a r g e s c a l e sucker, macrocheilus,  Catostomus  b r o w n c a t f i s h , I c t a l u r u s nebulosus, p r i c k l y s c u l p i n , Cottus  a s p e r , and h y b r i d s , M y l o c h e i l u s x R i c h a r d s o n i u s . D e v i l s Creek, a s m a l l s t r e a m 2-4 m e t e r s wide, was used to o b s e r v e spawning populations of M y l o c h e i l u s , R i c h a r d s o n i u s , and t h e i r hybrids. F l o w i n g into the l o w e r p o r t i o n of Stave L a k e f r o m D e v i l s Lake, D e v i l s C r e e k i s i d e a l l y suited f o r o b s e r v a t i o n because of its (1) s m a l l w a t e r volume, (2) good w a t e r c l a r i t y , and (3) short length (425 meters).  T h e s e three  f a c t o r s make o b s e r v a t i o n s p o s s i b l e at any time throughout i t s length.  Devils  C r e e k a l s o appears to be a m a j o r spawning a r e a i n the l o w e r p o r t i o n of Stave Lake.  8  • i o u r e 3.  Stave L a k e ,  British Columbia,  and adjacent w a t e r s .  9 Sampling F o u r r e g u l a r s a m p l i n g stations w e r e established, i n Stave L a k e to c o l l e c t adult M y l o c h e i l u s , R i c h a r d s o n i u s ,  and t h e i r h y b r i d s ( F i g . 3).  T h e s e w e r e a l l i n the l o w e r p o r t i o n of Stave Lake. was  H o w e v e r , some s a m p l i n g  done at the e x t r e m e n o r t h end (upper portion) of the lake and  w e r e a l s o abundant i n this l o c a l i t y .  hybrids  Samples w e r e taken by 19, 25, and  38mm  s t r e t c h m e s h m o n o f i l a m e n t n y l o n g i l l nets v a r y i n g i n s i z e f r o m 2. 4 x 15. 2 to 7. 6 x 15. 2 m.  In m o s t i n s t a n c e s , s a m p l i n g was  done at night since day-  t i m e s a m p l i n g was  v e r y ineffective.  adult M y l o c h e i l u s  w e r e s a m p l e d f r o m Babine, H a t z i c , A l o u e t t e , Ruby and  In a d d i t i o n to Stave Lake' s a m p l e s ,  O t t e r L a k e s and the Stave R i v e r , while R i c h a r d s o n i u s Babine, Shea, and Alouette Lakes. Richardsonius)  m  were sampled f r o m  No h y b r i d f i s h e s ( M y l o c h e i l u s x  are found i n any of these l o c a t i o n s .  Fingerling Mylocheilus, Richardsonius,  and t h e i r h y b r i d s were c o l l e c t e d  w i t h a fine m e s h dip net along the s h o r e s of the l o w e r p o r t i o n of Stave L a k e . A l l c o l l e c t i o n s of f i n g e r l i n g s w e r e made during daylight hours.  Heaviest  concentrations of f i n g e r l i n g s a p p e a r e d i n the l i t t o r a l a r e a s adjacent to D e v i l s and R o l l i e Creeks.  10 REPRODUCTIVE ISOLATION'BETWEEN MYLOCHEILUS AND RICHARDSONIUS Morphological Approach Two a p p r o a c h e s w e r e u s e d to d e t e r m i n e i f swamping i s o c c u r r i n g between M y l o c h e i l u s and R i c h a r d s o n i u s .  The f i r s t , and m o r e c l a s s i c a l  approach, attempts to m e a s u r e the p r e s e n c e of genes of one s p e c i e s i n populations of the other by m o r p h o l o g i c a l c h a r a c t e r c o m p a r i s o n s . S i g n i f i c a n t d i f f e r e n c e s i n m o r p h o l o g y between populations of e a c h s p e c i e s w i t h i n and outside the h y b r i d zone a r e taken as evidence f o r swamping. In Stave L a k e obvious h y b r i d s a r e excluded f r o m the c o m p a r i s o n s because they c a n not be c o n s i d e r e d as p e r m a n e n t m e m b e r s of e i t h e r species. A s e a r c h was made f o r m o r p h o l o g i c a l c h a r a c t e r s w h i c h a re w i d e l y d i v e r g e n t i n the two species.  Such c h a r a c t e r s a r e u s e f u l i n d e t e r m i n i n g  i f gene f l o w i s o c c u r r i n g between M y l o c h e i l u s and R i c h a r d s o n i u s .  The  c h a r a c t e r s u s e d i n this study are: 1)  a n a l f i n rays. Throughout its range, M y l o c h e i l u s t y p i c a l l y p o s s e s s 8 a n a l f i n rays.  Richardsonius' anal f i n ray number  isi quite v a r i a b l e throughout i t s range and d i f f e r e n t populations p o s s e s s d i f f e r e n t m e a n counts.  A c c o r d i n g to C a r l ,  Clemens,  and L i n d s e y (1959) m o s t B r i t i s h C o l u m b i a populations-possess a m e a n n u m b e r of 15 a n a l f i n rays.  In the h y b r i d zone, i n d i v i -  duals w h i c h r e s e m b l e t y p i c a l R i c h a r d s o n i u s p o s s e s s 14-22 a n a l f i n rays.  The mode i s e i t h e r 16 o r 17. A n a l f i n rays a r e not  used to detect swamping, however', but as a tool to separate obvious h y b r i d i n d i v i d u a l s f r o m m e m b e r s of the two p a r e n t a l species.  11 2)  L a t e r a l L i n e Scales.  Mylocheilus  p o s s e s s 68 to 79 r o w s of  l a t e r a l line s c a l e s while R i c h a r d s o n i u s  possess considerably  fewer, f r o m 54 to 67 rows ( C a r l , C l e m e n s , and L i n d s e y , 3)  1959).  R a t i o of P r e d o r s a l to P r e p e l v i c Length. The p o s i t i o n of the d o r s a l f i n r e l a t i v e to the p e l v i c f i n i s quite d i s t i n c t i n the two species.  In R i c h a r d s o n i u s , the d o r s a l f i n is i n s e r t e d c o n s i d e r -  a b l y p o s t e r i o r to the i n s e r t i o n of the p e l v i c fins while in M y l o c h e i l u s i t i s i n s e r t e d s o m e t i m e s i n line with, but m o r e often s l i g h t l y ahead of, the p e l v i c f i n i n s e r t i o n .  This r e l a t i o n -  ship i s q u a n t i f i e d as the ratio of the p r e d o r s a l to the p r e p e l v i c length. A l l counts and m e a s u r e m e n t s w i t h the exception of the p r e p e l v i c length are i n a c c o r d a n c e w i t h the p r o c e d u r e s suggested by Hubbs and L a g l e r (1958).  The p r e p e l v i c length i s the s t r a i g h t l i n e distance f r o m the  tip of the snout to the i n s e r t i o n of the o u t e r m o s t p e l v i c f i n ray. The d i s t r i b u t i o n s of l a t e r a l line s c a l e s and the p r e d o r s a l / p r e p e l v i c length r a t i o s a r e c o m p a r e d f o r populations of each s p e c i e s w i t h i n and outside the h y b r i d zone. Samples of M y l o c h e i l u s outside the h y b r i d zone w e r e c o l l e c t e d f r o m V a n c o u v e r Island and the Richardsonius  Sechelt P e n i n s u l a , w h i l e  w e r e s a m p l e d f r o m Alouette Lake.  In the c o m p a r i s o n s ,  obvious h y b r i d s are detected by t h e i r a n a l f i n r a y n u m b e r and Morphological-Biochemical The  excluded.  Approach  second approach, a c o m b i n e d m o r p h o l o g i c a l and b i o c h e m i c a l  attempts to m e a s u r e the frequency of v a r i o u s h y b r i d types.  one,  If swamping  12 i s o c c u r r i n g , b a c k c r o s s h y b r i d s should be m o r e abundant than T\  hybrids,  since m a n y generations of h y b r i d b a c k c r o s s e s should e x i s t i n the population. The  i n h e r i t a n c e of a n a l f i n rays a s c e r t a i n e d under l a b o r a t o r y conditions  a l l o w s a reasonable  s e p a r a t i o n of h y b r i d s into two groups; (1) F^  hybrids  and 1st generation h y b r i d b a c k c r o s s e s and (2) 2nd and l a t e r generation h y b r i d b a c k c r o s s e s and the p a r e n t a l species. and 1st generation h y b r i d b a c k c r o s s e s ,  The f o r m e r group, F\  i s separated  hybrids  into i t s component  p a r t s by two b i o c h e m i c a l c h a r a c t e r s , the i n h e r i t a n c e s of w h i c h a r e d e t e r m i n e d by e x p e r i m e n t a l c r o s s e s .  The  2nd and l a t e r generation h y b r i d b a c k -  c r o s s e s of the l a t t e r group are separated f r o m i n d i v i d u a l s of the p a r e n t a l s p e c i e s by these b i o c h e m i c a l c h a r a c t e r s , also.  The b i o c h e m i c a l  characters  used, w e r e chosen w i t h c a r e to include only p r o t e i n s w h i c h (1) show genetic v a r i a b i l i t y between --but not within--the two s p e c i e s , (2) p o s s e s s c o d o m i n ance between a l l e l e s and (3) a r e not s e x - l i n k e d .  These requirements insure  that any i n d i v i d u a l p o s s e s s i n g a l l e l e s of both species f o r the b i o c h e m i c a l c h a r a c t e r s i n question, i s detected.  13 Search for B i o c h e m i c a l  Characters  P o p u l a t i o n s of M y l o c h e i l u s and R i c h a r d s o n i u s  f r o m outside the  h y b r i d zone w e r e e x a m i n e d to detect b i o c h e m i c a l c h a r a c t e r s whose genetic c o n t r o l i s such that the l o c i i n the two s p e c i e s p o s s e s s homozygous, but different alleles.  E n z y m e s , e x a m i n e d and d i s c a r d e d because they e i t h e r  show no a l l e l i c d i f f e r e n c e s o r share a l l e l e s between the two s p e c i e s a r e e s t e r a s e s , p s e u d o c h o l i n e s t e r a s e s , l a c t i c dehydrogenase, s u c c i n i c dehydrogenase, i s o c i t r i c dehydrogenase, and g l u t a m i c dehydrogenase. Two p r o t e i n s , a g e n e r a l m u s c l e p r o t e i n , and an enzyme, m a l i c d e h y d r o genase show c o n s i s t e n t d i f f e r e n c e s between, but no v a r i a b i l i t y w i t h i n , the two species.  T h e s e c h a r a c t e r s a r e used to separate F^ h y b r i d s and 1st  generation hybrid backcrosses. Electrophoretic Analysis S a m p l e s of m u s c l e  are homogenized approximately  15 seconds i n  low i o n i c strength (0. 055M) phosphate b u f f e r , p H 7. 45, u n t i l the homgenate s p o s s e s s a c r e a m y texture.  The ratio  of m u s c l e to phosphate b u f f e r i s 1:2.  The homogenate i s then c e n t r i f u g e d f o r 15 m i n u t e s at 37, 000 x g and the supernatant c o l l e c t e d .  S t a r c h g e l e l e c t r o p h o r e s i s of the supernatant i s  conducted at 180 volts f o r 2. 25 h o u r s u s i n g a m i c r o method p r e v i o u s l y p u b l i s h e d ( T s u y u k i et a l . , 1966).  The g e n e r a l p r o t e i n zones a r e stained  w i t h a m i d o b l a c k 10B f o r three minutes.  M a l i c dehydrogenase i s stained  f o r two hours i n a solution containing (1) 23. 3 m l of 0. 1 M T r i s b u f f e r , pH 8. 5, (2) 1. 5 m l of 2 M m a l i c a c i d , (3) 0. 6 m l of 30 m g / r r i l n i c a t i n a m i d e adenine d i n u c l e o t i d e (NAD), (4) 0. 12 m l of 5 m g / m l phenazine methosulfate,  14  and  (5) 1.0  1963).  ml  of 10 m g / m l n i t r o b l u e t e t r a z o l i u m ( C o l o w i c k a n d  Kaplan,  15 R e a r i n g of E x p e r i m e n t a l  Animals  A r t i f i c i a l c r o s s e s of p a r e n t a l M y l o c h e i l u s , h y b r i d s , and  reciprocal hybrid backcrosses  Richardsonius,  reciprocal  to b o t h s p e c i e s w e r e m a d e  f r o m p a r e n t s t a k e n f r o m D e v i l s C r e e k i n 1966,  1967,  and  1968.  The  eggs w e r e kept i n a H e a t h i n c u b a t o r s u p p l i e d w i t h D e v i l s C r e e k water. The  w a t e r t e m p e r a t u r e i n the i n c u b a t o r w a s  the c r e e k . Artemia  Upon hatching,  the a m b i e n t t e m p e r a t u r e of  the f r y w e r e f e d a n i d e n t i c a l d i e t r e g i m e n of  n a u p l i i , l i v e plankton f r o m Stave L a k e , and f r o z e n adult b r i n e  s h r i m p u n t i l they r e a c h e d a n a l y s i s (35 mm). as parents  a s u f f i c i e n t s i z e to p e r m i t e l e c t r o p h o r e t i c  P r i o r to a n a l y s i s , a l l p r o g e n y a n d the w i l d f i s h u s e d  i n the 1967  and  1968  crosses were frozen.  c a r c a s s e s of the p a r e n t s u s e d i n the 1966 m a l i n which denatured  Unfortunately,  crosses were preseved  the  in for-  their proteins.  M y l o c h e i l u s and  Richardsonius  Outside  the H y b r i d  Zone  Muscle Proteins A t the c o n c e n t r a t i o n of the m u s c l e p r o t e i n e x t r a c t s t e s t e d , s e v e r a l c h a r a c t e r i s t i c m a j o r and m i n o r z o n e s a r e present.  The  muscle proteins  to be d i s c u s s e d a r e t h o s e m a j o r z o n e s a r b i t r a r i l y d e s i g n a t e d and  " 3 " ( F i g . 4).  "1",  A l l individual Mylocheilus  f r o m populations  the h y b r i d z o n e (Ruby, H a t z i c , A l o u e t t e , a n d  B a b i n e L a k e s and  d i s p l a y zone 3 w h e r e a s R i c h a r d s o n i u s ( A l o u e t t e , Shea, a n d  outside Stave  River)  f r o m o u t s i d e the h y b r i d zone e x c l u s i v e l y z o n e 1,  Babine L a k e s ) possess  of l e s s e r a n o d a l m o b i l i t y ( T a b l e I, F i g . 4). h y b r i d zone p o s s e s s b o t h z o n e s 1 a n d  "2",  3, o r  No 2.  a zone  i n d i v i d u a l s o u t s i d e the  16  ZONES-J.23  II |  | |  A. MYLOCHEILUS B. HYBRID C.HYBRID  •  *  1  W •  CATHODE  F i g u r e 4.  D. HYBRID E. RlCHARDSOK|»,lfi ANODE  S t a r c h g e l e l e c t r o p h o r e s i s of m u s c l e p r o t e i n s f r o m M y l o c h e i l u s c a u r i n u m , R i c h a r d s o n i u s balteatus, and t h e i r h y b r i d s . S a m p l e s a r e f r o m Stave L a k e , B r i t i s h Columbia.  T A B L E I.  M u s c l e p r o t e i n and m a l i c dehydrogenase patterns of M y l o c h e i l u s and R i c h a r d s o n i u s outside the h y b r i d zone (number of i n d i v i d u a l s ) . "M" = M y l o c h e i l u s ; "R" = R i c h a r d s o n i u s .  M a l i c Dehydrogenase  Muscle Proteins Area  Species  Zone Zones 1 1,2  Zones 1,2,3  Zones 2,3  Zone 3  Zone 3(5)  Zones Zones 1,2, 3 12345  Zones Zones 3,4,5 1,3,(5'  Ruby L a k e , B.C.  M  35  35  Stave R i v e r , B.C.  M  31  31  H a t z i c L a k e , B.C.  M  25  25  Alouette L a k e , B.C.  M  27  38  Babine L a k e , B.C.  M  10  Shea L a k e , B.C.  R  25  25  Alouette L a k e , B.C.  R  36  53  Babine L a k e , B.C.  R  13  18 Malic  Dehydrogenase  P o p u l a t i o n s of R i c h a r d s o n i u s exhibit a m a l i c d e h y d r o g e n a s e p a t t e r n with three isozymes,  d e s i g n a t e d z o n e "1", "3", a n d " 5 " ( F i g . 5 B ) .  v a r i a b i l i t y i s s e e n i n the two p o p u l a t i o n s e x a m i n e d : L a k e s ( T a b l e I)-  MostMylocheilus  No  Shea a n d Alouette  p o s s e s s a single M D H  isozyme  with  a m o b i l i t y v e r y s i m i l a r to that of the " 3 " i s o z y m e i n R i c h a r d s o n i u s . It i s a l s o d e s i g n a t e d a s zone " 3 " ( F i g . 5 A , F ) . the z o n e 3 i s o z y m e i n M y l o c h e i l u s  In s o m e i n s t a n c e s , h o w e v e r ,  continues weakly f o r some distance  a n o d a l l y a n d o f t e n t e r m i n a t e s i n w h a t a p p e a r s to b e a s e c o n d z o n e c o r r e s p o n d i n g to that of z o n e 5 i n R i c h a r d s o n i u s p r e s e n t i n M y l o che i l u s  ( F i g . 5G).  unless a considerable " s l u r " is also present,  s u g g e s t i n g that it m a y be a n a r t e f a c t o f the t e c h n i q u e . accorded  T h i s zone i s n e v e r  It i s not, t h e r e f o r e ,  m u c h w e i g h t i n the s u b s e q u e n t a n a l y s i s o f h y b r i d i n d i v i d u a l s .  I n h e r i t a n c e of B i o c h e m i c a l  Characters  Muscle Proteins C r o s s e s of M y l o c h e i l u s x M y l o c h e i l u s a n d R i c h a r d s o n i u s  x  Richardsonius  r e s u l t i n p r o g e n y w i t h p r o t e i n p a t t e r n s c h a r a c t e r i s t i c of the r e s p e c t i v e parents  ( T a b l e II).  The F ^ progeny resulting f r o m r e c i p r o c a l c r o s s e s of  M y l o c h e i l u s and Richardsonius  produce a single e l e c t r o p h o r e t i c pattern  d i s p l a y i n g z o n e s 1 a n d 3 i n a d d i t i o n to a u n i q u e i n t e r m e d i a t e zone, d e s i g nated "2" ( F i g . 4C). to M y l o c h e i l u s  of p r e s u m e d Fj^ h y b r i d s  ( B G M ) ^ p r o d u c e o f f s p r i n g with three types of p r o t e i n  1) F o r s i m p l i c i t y , Mylocheilus,  Reciprocal backcrosses  s o m e a b b r e v i a t i o n s a r e u s e d i n t h e text.'  "R" = R i c h a r d s o n i u s ,  Mylocheilus x Richardsonius F j hybrid backcross Richardsonius. '  T h e y a r e : "M" = '  "F]_" = individual resulting  c r o s s unless stated otherwise,  from  "BCM" =  to M y l o c h e i l u s , " B C R " = F ^ h y b r i d b a c k c r o s s to  19  ZONES  I 2 3 4(5) A. MYLOCHEII US B. RICHARDSONIUS C. HYBRID •.HYBRID E. HYBRID  ZONES  3  (5) F. MYLOCHEILUS G. MYLOCHEILUS  F i g u r e 5.  S t a r c h - g e l e l e c t r o p h o r e s i s of m a l i c d e h y d r o g e n a s e f r o m M y l o c h e i l u s c a u r i n u m , R i c h a r d s o n i u s balteatus, and t h e i r hyb r i d s .  T A B L E II.  M u s c l e protein patterns  of p a r e n t s a n d p r o g e n y of c r o s s e s m a d e i n 1966 a n d 1967.  PROTEIN  PATTERNS P r o g e n y (No. of i n d i v i d u a l s )  Parents Zone T y p e of C r o s s Female  Male  Mylocheilus  Mylocheilus  Mylocheilus  "Hybrid"  "Hybrid"  Mylocheilus  Mylocheilus  Richardsonius  R i c h a r d s onius  Mylocheilus  R i c h a r d s onius  "Hybrid"  R i c h a r d s onius  "Hybrid"  "Hybrid"  R i c h a r d s onius  Richardsonius  R i c h a r d s onius  Female 1 2 3  + +  +  Zone Numbers  Numbers Male 1 2 3  +  +  + +  1  2  3 +  1 2 +  3  1 2  3  1 2  +  +  +  +  +  +  40  +  4  5  3  +  6  22  14  +  +  1 2 +  40 4  +  +  a* +  3  40  +  +  +  1 7  +  y p a r e n t a l p a t t e r n s f r o m 1966 c r o s s e s n o t a v a i l a b l e .  5 1 9  5 33  .3  21  patterns:  a) the p a r e n t a l M y l o c h e i l u s  possessing  z o n e s 1, 2, 3, a n d  3 and corresponding presumed  F^ hybrids  1,  to R i c h a r d s o n i u s  b) the p a t t e r n  possessing  z o n e 3,  b) the p a t t e r n  c) a n e w p a t t e r n p o s s e s s i n g  to that of F i g u r e 4 B .  three types of p r o t e i n p a t t e r n s : zone  pattern,  only zones 2 and  R e c i p r o c a l b a c k c r o s s e s of  (BCR) also produce progeny  a) the p a r e n t a l R i c h a r d s o n i u s  possessing  with  pattern,  z o n e s 1, 2, a n d 3 a n d c) a n e w p a t t e r n  only zones 1 and 2 and c o r r e s p o n d i n g  to that o f F i g . 4D.  A g e n e t i c m o d e l e x p l a i n i n g the i n h e r i t a n c e o f t h e s e p r o t e i n s i s d e p i c t e d i n F i g u r e 6. two l o c i ,  T h e m o d e l p r o p o s e s that z o n e s  1 and 3 a r e each controlled by  "A", a n d "B", e a c h of w h i c h e x i s t s i n two a l l e l i c f o r m s . , M y l o c h e i l u s  i s d e p i c t e d a s b e i n g h o m o z y g o u s f o r the g e n o t y p e , A B • a n d A h o m o z y g o u s f o r A'B';  Richardsonius  B  W h i l e the m o d e l d e p i c t s l o c i A a n d B a s b e i n g o n  A'B' separate  chromosomes,  they may,- i n f a c t , be l o c a t e d on the s a m e  s o m e b u t a t l e a s t 50 m a p u n i t s a p a r t . A s s u m p t i o n s w h i c h a r e i m p l i c i t i n the m o d e l a r e : 1)  P r o t e i n s detected as zones 1 and 3 a r e c o n t r o l l e d by genes present i n both parental species.  2)  T h e p r o d u c t s of the l o c i . c o m p l e x  to f o r m a d i m e r  which  i s u n i q u e to e a c h p a r e n t a l s p e c i e s . 3)  P r o t e i n zone 2 r e p r e s e n t s polypeptide  4)  a h y b r i d d i m e r c o m p o s e d of a  c o n t r i b u t e d f r o m e a c h of the p a r e n t a l s p e c i e s .  The polypeptides  o f p r o t e i n zone  1 are electrophoretically  i n d i s t i n g u i s h a b l e a s a r e t h o s e o f p r o t e i n z o n e 3.  chromo-  MYLOCHEILUS GENOTYPE  PROTEIN  Bl  ZONES.  RICHARDSONIUS  IB  Bl  2 3  I  IB 2 3  F, HYBRID  tsJ  ro  BACKCROSSES TO MYLOCHEILUS A|  |A  Bl  IB 2 3  F i g u r e 6.  1|  A  A Bl I  IB 2 3  Bl  IB 2 3  B I  B 2 3  B,  BACKCROSSES T O RICHARDSONIUS  B. 2 3  A g e n e t i c m o d e l f o r the i n h e r i t a n c e o f m u s c l e p r o t e i n s , and Myfcooheilus, r e s p e c t i v e l y .  A  A  A  B'  B*  B  2 3  I  2 3  z o n e s 1 a n d 3, i n  B  (  B' 2 3  Richardsonius  23 The true breeding quality of artificial parental crosses is evidence for homozygosity of alleles A and B in Mylocheilus  and A' and B' in  Richardsonius. Further evidence is the lack of variation in these protein zones in either species outside the hybrid zone.  The distribution of protein  patterns in F j hybrid backcrosses to both species supports the idea of dual gene control.  For example, in F^ hybrid backcrosses to Mylocheilus  a new type of pattern in found in addition to the expected parental (zone 3) and F^ (zones 1,2,.3) patterns.  This pattern contains zones 2 and 3 but  not zone 1 (Fig. 4B). If zone 2 is composed of subunits of zones 1 and 3, its presence indicates that only half of the components required to produce zone 1 are present.  The half of the components of zone 1 which is present  in this phenotype combines with some of the components of zone 3 to produce zone 2. Thus, only half of the genetic complement of Richardsonius is present in these phenotypes; the other half has segregated to another gamete.  In presumed F i hybrid backcrosses to Richardsonius a similar  situation occurs in which the new pattern possesses only zones 1 and 2 (Fig. 4D), indicating that only half of the components needed to produce the Mylocheilus  zone 3 is present.  In analogous fashion to the two-zoned  BGM progeny, the components of zone 3 which are present combine with some of those of zone 1 to form zone 2. If the model is correct, the F i hybrid backcross progeny to both species should possess one-zoned, two-zoned, and three-zoned patterns in a ratio of 1;2;1, respectively. This hypothesis is not rejected for either BCM progeny (X = 1. 81) or BCR 2  progeny (X =z. 75 < X 2  2  •_ 5, df =2 ' =5  0  Although observed,  the s e x e s of a r t i f i c i a l l y p r o d u c e d F.j h y b r i d s w e r e not  t h e r e i s no e v i d e n c e  that the p r o t e i n zones' a r e c o n t r o l l e d  by  s e x - l i n k e d genes, f o r r e c i p r o c a l F^  c r o s s e s p r o d u c e d o n l y one p r o t e i n  p a t t e r n , n a m e l y , z o n e s 1,2,  A  these c r o s s e s would  3.  d i s c r e p a n c y i n the s e x r a t i o s of  not l i k e l y p r o d u c e the a b o v e r e s u l t s i n c e the  r a t i o of n a t u r a l h y b r i d s The  and  do  not d i f f e r s i g n i f i c a n t l y f r o m  s t u d y of p r o g e n y p r o d u c e d u n d e r e x p e r i m e n t a l  1:1  sex  (See p a g e 71 ).  conditions  clari-  f i e s the t y p e ( s ) of i n d i v i d u a l ( h y b r i d , p a r e n t a l , etc. ) a s s o c i a t e d w i t h different m u s c l e protein patterns.  F o r example, individuals which  only zone 1 m a y  e i t h e r be p u r e R i c h a r d s o n i u s  Richardsonius  while,  be p u r e M y l o c h e i l u s  similarly,  or hybrid backcrosses  those w h i c h p o s s e s s  or h y b r i d b a c k c r o s s e s  zone 3 m a y  to M y l o c h e i l u s .  The  three-  b y the F j h y b r i d b a c k c r o s s e s  e i t h e r s p e c i e s o r the F ^ h y b r i d .  two  to  t y p e s of t w o - z o n e d p r o g e n y ,  z o n e s 1, 2 a n d  z o n e s 2, 3, a r e p o s s e s s e d  Richardsonius  and M y l o c h e i l u s , r e s p e c t i v e l y .  Any  to  either  z o n e d p a t t e r n ( F i g . 4, C) i s p o s s e s s e d The  possess  only by h y b r i d b a c k c r o s s e s  of the a b o v e m u s c l e p r o t e i n patterns- c o u l d be p o s s e s s e d  h y b r i d s a c c o r d i n g to the g e n e t i c m o d e l p r e s e n t e d above.  by  to  F^  However, it is  a s s u m e d that t h e y do not e x i s t n a t u r a l l y a s e x p e r i m e n t a l l y p r o d u c e d o n e s a r e extremely  inviable w h e r e a s p a r e n t a l s p e c i e s ' p r o g e n y and  r a i s e d , u s u a l l y without  difficulty.  F j hybrids were  25 M a l i c Dehydrogenase  (MDH)  P a r e n t a l c r o s s e s of M y l o c h e i l u s x M y l o c h e i l u s a n d Richardsonius  yield progeny which possess  c h a r a c t e r i s t i c of the r e s p e c t i v e p a r e n t s reciprocal F j crosses possess Four MDH  m a l i c dehydrogenase patterns  ( T a b l e III, F i g . 5).  P r o g e n y of  w h e n p r o g e n y of r e c i p r o c a l p r e s u m e d  to M y l o c h e i l u s  are examined.  They are:  the p a r e n t a l M y l o c h e i l u s p a t t e r n , b) z o n e s 1,2,3,4, (5), the F ^ pattern, two  a n d d) z o n e s 3,4(5) ( F i g . 5).  progeny f r o m hybrid backcrosses  1967. and  c) z o n e s 1,2,3  One  possessed  the o t h e r ,  to R i c h a r d s o n i u s  the t y p i c a l R i c h a r d s o n i u s  u n c e r t a i n t y r e g a r d i n g the e x i s t e n c e of M D H  quaternary  to R i c h a r d s o n i u s ,  Fj  First,  only  were reared in  p a t t e r n , z o n e s 1, 3,  (5)  B e c a u s e of the  zone 5 i n M y l o c h e i l u s , the l a c k  and  the u n c e r t a i n n a t u r e  of  MDH  Additionally,  However,  s e v e r a l a s p e c t s of the i n h e r i t a n c e a r e  c o d o m i n a n c e e x i s t s f o r the a l l e l e s c o n t r o l l i n g M D H  hybrids possess  the z o n e s of b o t h M y l o c h e i l u s a n d  two new  since  Richardsonius.  z o n e s , z o n e s 2 a n d 4 a r e f o u n d i n the F ^  n e i t h e r i s p r e s e n t i n the p a r e n t a l s p e c i e s . hybrid MDH  hybrid  s t r u c t u r e i n o t h e r o r g a n i s m s , a d e t a i l e d g e n e t i c a n a l y s i s of t h i s  e n z y m e i s not a t t e m p t e d . apparent.  F^  a) z o n e 3,  Unfortunately,  the F ^ h y b r i d p a t t e r n , z o n e s 1,2,3,4, (5).  of h y b r i d b a c k c r o s s e s  x  a s i n g l e p a t t e r n w i t h z o n e s 1,2,3,4,(5),  patterns are observed  hybrid backcrosses  Richardsonius  Presumably,  hybrids,  these new  zones are  i s o z y m e s f o r m e d by the u n i o n of s u b u n i t s f r o m n o r m a l l y  ring isozymes  i n the two  species.  S e c o n d , t h e r e m u s t be at l e a s t two  o r g r o u p s of l o c i , ' c o n t r o l l i n g the s y n t h e s i s of M D H  in these  but  occurloci,  species since  T A B L E III.  M a l i c d e h y d r o g e n a s e p a t t e r n s of p a r e n t s a n d p r o g e n y of c r o s s e s m a d e i n 1967.  Malic  Dehydrogenase  Parents Zone Male  1 2 3 4 (5)  Mylocheilus  +  Mylocheilus  Hybrid**  +  Hybrid**  Mylocheilus  Mylocheilus  Richardsonius  Richardsonius  Mylocheilus  +  +  Richardsonius  Hybrid**  +  +  + +++  Richardsonius  Richardsonius  +  +  +  F  x  hybrid  Numbers  Male  1 2 3 4(5)  Mylocheilus  ** p r e s u m e d  Zone  Numbers  Female  T y p e of C r o s s Female  Patterns Progeny  + + +++  + +++  + +  +  •  3 (5) 1 2 3  3 4 (5)  1.2 3 4 ( 5 )  40 12  11  14  16  7  7  11  17  +  35  +  40  +  1 3 (5)  1  1 33  27 f o u r p h e n o t y p e s a r e o b s e r v e d i n the p r e s u m e d F ^ h y b r i d b a c k c r o s s e s to M y l o c h e i l u s . If a s i n g l e l o c u s i s i n v o l v e d , o n l y the p a r e n t a l M y l o c h e i l u s a n d the F ^ h y b r i d M D H of M D H  p a t t e r n s s h o u l d be p r o d u c e d .  i n these b a c k c r o s s e s compose 4 4 %  Non-parental  of a l l progeny.  The  types  hypothesis  that the r a t i o of p a r e n t a l to n o n - p a r e n t a l t y p e s i n - F ^ h y b r i d b a c k c r o s s e s to Mylocheilus  i s not s i g n i f i c a n t l y d i f f e r e n t f r o m  1:1 i s not r e j e c t e d (  =  2 1. 25 <  a •  =  05,  df=l  =  ^- 84).  If i n f a c t , the h y p o t h e s i s i s t r u e , the  two  l o c i o r g r o u p s of l o c i a r e a s s o r t i n g c o m p l e t e l y independently. A s w i t h the e x p l a n a t i o n of m u s c l e p r o t e i n i n h e r i t a n c e , the sex was determined  f o r the F ^ h y b r i d p r o g e n y .  However,  not  sex-linked inheritance is  not i n d i c a t e d s i n c e r e c i p r o c a l F j h y b r i d s b o t h p r o d u c e  one a n d the s a m e  pattern. The  s t u d y of p r o g e n y p r o d u c e d  under experimental condition  the t y p e ( s ) of i n d i v i d u a l s a s s o c i a t e d w i t h the v a r i o u s M D H  clarifies  patterns.  w h i c h p o s s e s s the p a t t e r n , z o n e s 3 (5) c a n be p u r e M y l o c h e i l u s  Those  or hybrid  b a c k c r o s s e s to M y l o c h e i l u s w h i l e t h o s e w h i c h p o s s e s s the z o n e s 1,3,(5) p a t t e r n c a n e i t h e r be p u r e R i c h a r d s o n i u s o r h y b r i d b a c k c r o s s e s to Richardsonius.  The  z o n e s 1, 2, 3, 4, (5) M D H  pattern is possessed  by  r e c i p r o c a l F ^ h y b r i d s a n d p r e s u m e d F j h y b r i d b a c k c r o s s e s to e i t h e r s p e c i e s . The M D H  p a t t e r n s , z o n e s 1, 2, 3 a n d z o n e s 3,4, (5) a r e u n i q u e to h y b r i d  b a c k c r o s s e s to M y l o c h e i l u s .  H o w e v e r , i t i s p o s s i b l e that they c o u l d be  p o s s e s s e d by h y b r i d b a c k c r o s s e s to R i c h a r d s o n i u s ;  i t i s not e l u c i d a t e d  b e c a u s e o n l y 2 i n d i v i d u a l h y b r i d b a c k c r o s s e s to R i c h a r d s o n i u s duced experimentally in  1967.  were pro-  28 Linkage If two b i o c h e m i c a l c h a r a c t e r s a r e to be t w i c e a s u s e f u l a s a s i n g l e one  i n d e t e c t i n g h y b r i d s they m u s t be i n d e p e n d e n t o f one a n o t h e r ;  m u s t n o t be t i g h t l y l i n k e d .  they  A c h i - s q u a r e t e s t o f i n d e p e n d e n c e o f the two  p r o t e i n s w a s p e r f o r m e d a n d the h y p o t h e s i s that they a r e i n d e p e n d e n t i s rejected  (X  2  = 22. 5.9>  X  2  _ Q Q  d a t a ( T a b l e I V ) r e v e a l s that M D H  5  ^  _ ^ = 12. 59).  A n i n s p e c t i o n o f the  p a t t e r n , z o n e s 1,2,3,4,(5),  occurs  m u c h t o o f r e q u e n t l y w i t h m u s c l e p r o t e i n p a t t e r n , z o n e s 2, 3, than, w o u l d be expected  i f the two p r o t e i n s a r e n o t l i n k e d .  the s i g n i f i c a n t c h i - s q u a r e v a l u e . are partially linked, drastically reduced characters.  Although  T h i s cell(#6) c o n t r i b u t e s 4 4 % of i t a p p e a r s that the two p r o t e i n s  t h e i r c o m b i n e d value i n d e t e c t i n g h y b r i d s i s not w h e n c o m p a r e d to two c o m p l e t e l y  independent o r unlinked  F o r e x a m p l e , the p r o b a b i l i t y o f r e c o g n i z i n g p r e s u m e d F ^  hybrid backcrosses  to M y l o c h e i l u s  f r o m t h o s e c r o s s e s s h o w n i n T a b l e IV,  a s s u m i n g i n d e p e n d e n c e , i s p ( a l l c e l l s ) - p ( c e l l 1 + c e l l 10) = 0. 875. d u a l s o f c e l l s 1 a n d 10 a r e c l a s s i f i e d a s p u r e M y l o c h e i l u s respectively.  Indivi-  and F^ hybrids,  T h e p r o p o r t i o n o f p r e s u m e d F ^ h y b r i d b a c k c r o s s e s to  Mylocheilus actually observed the t h e o r e t i c a l v a l u e .  i s 0. 852, a d e c r e a s e  o f o n l y 0. 023 f r o m  29  TABLE  IV.  C o r r e l a t i o n of m a l i c d e h y d r o g e n a s e , a n d m u s c l e p r o t e i n p a t t e r n s of h y b r i d b a c k c r o s s e s to M y l o c h e i l u s .  Numbers  in parentheses are expected proportions f o r each c e l l a s s u m i n g i n d e p e n d e n c e of i n h e r i t a n c e of the two p r o t e i n s . E n c i r c l e d n u m b e r s a r e p a r t i c u l a r c e l l s r e f e r r e d to i n the text. Malic Zones  oteins  3  2  3  3  (5)  ©  1 d/16)  2  ©  scle  0/8) 1  2  3  ©  4 (1/16)  Dehydrogenase  1 2  3  3 .  4  (5)  d/16)  (5)  ©  3 (1/16)  © 5 d/8)  (1/8)  7  4  ©  d/16)  15  3  ©  5  ©  @  (1/16)  1 2  3  3 (1/16)  5  ©  ©  d/8) j (1/16)  ©  30  .  . .  I n h e r i t a n c e of A n a l F i n Rays;. S e p a r a t i o n of 2nd a n d Generation Hybrid The  s e p a r a t i o n of 2nd a n d  later generation hybrid backcrosses  1st g e n e r a t i o n h y b r i d b a c k c r o s s e s estimate  of t h e i r a b u n d a n c e and,  advantage or disadvantage  n  and  F , r  Evidence  hybrids permits of the  an  selective  In t h i s  study,  supporting this a s s u m p -  e g g s u s e d i n F ^ x F i c r o s s e s i n 1966, mm).  only 4  In e s s e n c e ,  the  to the s e p a r a t i o n of 1st g e n e r a t i o n h y b r i d b a c k c r o s s e s  and  F j hybrids f r o m a l l subsequent hybrid b a c k c r o s s The  from  g r o u p s i s s i m p l i f i e d by a s s u m i n g that  i n d i v i d u a l s w e r e r e a r e d to the f i n g e r l i n g s t a g e (^60 task is reduced  n  of v a r i o u s h y b r i d g e n e r a t i o n s .  h y b r i d s do not e x i s t i n n a t u r e .  t i o n i s the f a c t that of 632  F;?,. • • F  i n d i r e c t l y , an e s t i m a t e  the d i f f i c u l t y of s e p a r a t i n g the two F£- • • F  Later  Backcrosses  generations.  i n h e r i t a n c e of a t h i r d c h a r a c t e r , a n a l f i n r a y s , was  a t t e m p t to e f f e c t a s e p a r a t i o n of t h e s e two  groups.  studied in an  A n a l fin rays were  c h o s e n a s the c h a r a c t e r b e c a u s e 1) t h e y a r e w i d e l y d i v e r g e n t i n the species; usually 8 in number in M y l o c h e i l u s ,  14-22  in Richardsonius  two and  2)  they d i s p l a y a p o l y g e n e t i c i n h e r i t a n c e w i t h no a p p a r e n t d o m i n a n c e ( F i g . 7). E x p e r i m e n t a l l y produced F^ hybrids possess with approximately hybrids possess Backcrosses  93%  possessing either  9(4. 2 % )  o r 12(2. 1 % )  f r o m 9 to 12 a n a l f i n r a y s  10 o r 11 ( F i g . 7).  V e r y few  Fj  anal fin rays.  of p r e s u m e d F ^ h y b r i d s w i t h 10,  11, a n d  12 a n a l f i n r a y s ,  to M y l o c h e i l u s , m a i n l y w i t h 8, y i e l d p r o g e n y w i t h 7 to 11 a n a l f i n r a y s , w i t h a l a r g e m o d e at 9(65. 9 % ) . A  S m a l l e r p e a k s a r e at 8(15. 2 % )  c r o s s of a h y b r i d m a l e w i t h 9 a n a l f i n r a y s ( d e t e r m i n e d  1st g e n e r a t i o n h y b r i d b a c k c r o s s and a f e m a l e M y l o c h e i l u s  to M y l o c h e i l u s  but  a n d at 10(17.1%).  to be at l e a s t a  on the b a s i s of i t s p r o t e i n s )  {8 r a y s ) p r o d u c e d p r o g e n y w i t h  mainly  31  PARENTS .  RXR  15 16 17 18 19  N-153  ? 1 i 2  i  <?  1 3  I  3  PARENTS  * 1st B C R  N - 75  PARENTS  F, HYBRIDS N = 305  7 8 14 15 16 17 l( MJ2~22~  PARENTS  s 1st B C M N - 178  7 8 10 I! 12 20  "M H  * 2nd B C M  14 2  PARENTS  N =40 H  8  9  I  !  P A R E N T S 7J3  <?[ 15 10  ANAL F i g u r e 7.  19  !  FIN  RAYS  20  J1V1BERS  A n a l t i n r a y d i x t r i bu.Lion of ].9b6 arid 19o7 e x p e r i m e n t a l c r o s s e s . c o m b i n e d . P a r e n t s of c r o s s e s i n c l u d e d on r i o h t s ide of g ra ph.  32 8 anal fin rays 9,  ( 8 0 % ). O f the r e m a i n i n g  a n d 2. 5 % p o s s e s s e d  2nd  10 a n a l f i n r a y s .  generation hybrid backcrosses  progeny  17. 5 %  possessed  T h e s e p r o g e n y w e r e at l e a s t  to M y l o c h e i l u s .  Presumed F^ hybrid backcrosses  to the o t h e r  species,  y i e l d p r o g e n y w i t h a n a l f i n r a y s r a n g i n g f r o m 11 to 15. i n d i v i d u a l s w i t h 12 to 14 a n a l r a y s c o m p o s e  Richardsonius,  However,  8 4 % o f the B C R  U n f o r t u n a t e l y , no s e c o n d g e n e r a t i o n B C R s w e r e p r o d u c e d  those  progeny.  experimentally.  O n the b a s i s o f a n a l f i n r a y i n h e r i t a n c e , 8 a n a l f i n r a y s w e r e  chosen  a s the d i v i d i n g l i n e ; t h o s e w i t h 8 o r f e w e r a r e c o n s i d e r e d to be e i t h e r p u r e Mylocheilus  o r at l e a s t 2nd g e n e r a t i o n  hybrid backcrosses  to M y l o c h e i l u s ,  w h i l e those w i t h 9 to 11 a n a l r a y s a r e c o n s i d e r e d to be e i t h e r 1st g e n e r a t i o n B C M s o r F j hybrids.  However,  i f the d i s t r i b u t i o n s f o r v a r i o u s  s h o w n i n F i g u r e 7 a r e r e p r e s e n t a t i v e o f those i n n a t u r e , 16%  crosses  approximately  o f the 1st g e n e r a t i o n B C M s w o u l d be c l a s s i f i e d i n the w r o n g  category;  t h e y w o u l d be c l a s s e d a s a t l e a s t 2nd g e n e r a t i o n B C M s , t h e r e f o r e , p r o d u c ing an o v e r e s t i m a t e  o f the l a t t e r .  O n the o t h e r hand, a p p r o x i m a t e l y  20%  of the 2nd g e n e r a t i o n B C M s w o u l d be c l a s s e d a s p a r t o f the p o p u l a t i o n c o n t a i n i n g 1st g e n e r a t i o n B C M s a n d F j h y b r i d s .  Such s o u r c e s of e r r o r  cannot  be e l i m i n a t e d f r o m the m e t h o d . F o r hybrid backcrosses  to R i c h a r d s o n i u s ,  15 a n a l f i n r a y s w e r e chosen'  a s the d i v i d i n g l i n e ; t h o s e w i t h 15 o r g r e a t e r a r e c l a s s e d a s at l e a s t 2nd g e n e r a t i o n B C R s o r p u r e R i c h a r d s o n i u s , w h i l e t h o s e w i t h 11 to 14 a n a l r a y s a r e c l a s s e d a s 1st g e n e r a t i o n B C R s o r F ^ h y b r i d s .  Approximately  4<% of the f i r s t g e n e r a t i o n B C R s w o u l d be m i s c l a s s i f i e d a s 2nd g e n e r a t i o n o r l a t e r B C R s u n d e r this scheme.  Unfortunately,  2nd g e n e r a t i o n  BCRs  w e r e not p r o d u c e d and c o n s e q u e n t l y no e s t i m a t e c o u l d be m a d e of the i n d i v i d u a l s w h i c h w o u l d be m i s c l a s s i f e d a s Linkage An  1 st g e n e r a t i o n B C R s .  of B i o c h e m i c a l a n d M o r p h o l o g i c a l  important  hybrid backcross  Characters  c o n s i d e r a t i o n i n e s t i m a t i n g the f r e q u e n c y t y p e s i s the n o n - l i n k a g e  with anal fin rays.  Otherwise,  rays always possess  of v a r i o u s  of e i t h e r b i o c h e m i c a l c h a r a c t e r  an e r r o n e o u s  F o r e x a m p l e , i f those h y b r i d b a c k c r o s s e s  estimate may  to M y l o c h e i l u s  the p a r e n t a l M y l o c h e i l u s  be  obtained.  with 8 anal fin  m u s c l e p r o t e i n and  d e h y d r o g e n a s e p a t t e r n s r a t h e r than h y b r i d p a t t e r n s , m o s t 2nd and generation B C M s may  not be d e t e c t e d , e v e n w h e n p r e s e n t .  protein or MDH  patterns are investigated.  to M y l o c h e i l u s  w e r e s e g r e g a t e d into g r o u p s of 7+8  The  w i t h 7+8 v rays ( x  X  later  muscle  backcrosses and 9 to 12 a n a l f i n  p r o p o r t i o n of v a r i o u s p a t t e r n s of m u s c l e p r o t e i n a n d M D H  then c o m p a r e d f o r the two V a n d VI,  Hybrid  malic  Consequently,  p o s s i b l e l i n k a g e r e l a t i o n s h i p s between a n a l f i n r a y n u m b e r and  rays.  •  groupings.  The  data, p r e s e n t e d i n T a b l e s  s h o w that f o r b o t h m u s c l e p r o t e i n a n d M D H  a n a l f i n r a y s do not d i f f e r =1  2  muscle proteins  05 < X ^  are  patterns, individuals  s i g n i f i c a n t l y f r o m t h o s e w i t h 9 to 12 2  a  = . 05,  df = 2  =5  y y  QQ'  A  x"  2  MDH  ~ 3.  39<  =7.81)  2  a  =. 05,  df = 3  .  Linkage  f i n r a y s i s , t h e r e f o r e , not  of the two b i o c h e m i c a l c h a r a c t e r s w i t h a n a l  found.  U n f o r t u n a t e l y , i n s u f f i c i e n t n u m b e r s of p r o g e n y w e r e r e a r e d a n d y s e d to p e r m i t biochemical  anal-  s u c h a d e t e r m i n a t i o n of l i n k a g e r e l a t i o n s h i p s b e t w e e n  and m o r p h o l o g i c a l c h a r a c t e r s f o r h y b r i d b a c k c r o s s e s  to  34  T A B L E V.  T h e p r o p o r t i o n of v a r i o u s m u s c l e p r o t e i n p a t t e r n s of presumed F  x  hybrid backcrosses  r e l a t i o n to anal f i n r a y number.  to M y l o c h e i l u s i n Numbers  in parantheses  a r e e x p e c t e d v a l u e s a s s u m i n g h o m o g e n i t y of the two distributions.  Muscle Protein Zones  1  Anal F i n Rays _ ' 7 + 8 9 t o 12  2  3  4(3.53)  13(13.47)  2  3  4(5.40)  22(20.60)  3  3(2.08)  7(7.92)  35  T A B L E VI.  The proportion  of v a r i o u s m a l i c d e h y d r o g e n a s e p a t t e r n s  of p r e s u m e d F  hybrid backcrosses  x  r e l a t i o n to a n a l f i n r a y n u m b e r . are expected  to M y l o c h e i l u s i n  Numbers i n parentheses  v a l u e s a s s u m i n g h o m o g e n i t y of the two  distributions.  Anal F i n Rays MDH  Zones  1  3  2  1 2  4 (5)  3  7+ 8  9 to 12  3(5.19)  22(19.81)  2(1.66)  6(6.34)  3  4 (5)  3(2.70)  10(10.30)  3  (5)  3(1.45)  4(5.55)  a  36 Richardsonius.  A s a result,  the a s s u m p t i o n h a s to be m a d e that the  same linkage relationships hold for R i c h a r d s o n i u s  a s they do f o r  Mylocheilus.  S e p a r a t i o n of F ^ H y b r i d s a n d 1st G e n e r a t i o n s  Hybrid  Backcrosses  B a s e d o n the i n h e r i t a n c e of a n a l f i n r a y s d i s c u s s e d i n a p r i o r s e c t i o n , a n a t t e m p t i s m a d e to s e p a r a t e 2 n d a n d l a t e r g e n e r a t i o n h y b r i d b a c k crosses from section,  and F^ hybrids.  In this  s e p a r a t i o n o f the F ^ h y b r i d s f r o m the 1st g e n e r a t i o n h y b r i d  backcrosses and M D H  1st g e n e r a t i o n h y b r i d b a c k c r o s s e s  i s b a s e d o n the k n o w l e d g e of the i n h e r i t a n c e of m u s c l e p r o t e i n  patterns.  In c e r t a i n c a s e s ,  hybrid backcrosses  c a n b e i d e n t i f i e d f r o m the  a n a l y s i s o f a s i n g l e p r o t e i n b e c a u s e o f the u n i q u e n e s s of c e r t a i n p a t t e r n s (Fig.  8C). H o w e v e r , d e t e c t i o n o f m a n y h y b r i d b a c k c r o s s e s  an examination  o f the two p r o t e i n s s i m u l t a n e o u s l y  Mylocheilus M D H origin;  pattern.  to M y l o c h e i l u s  the F ^ h y b r i d m u s c l e p r o t e i n p a t t e r n but the  pattern.  The muscle protein pattern indicates its hybrid  the M y l o c h e i l u s M D H  a F i h y b r i d since a l l  from  ( F i g . 8). F o r e x a m p l e ,  f i s h "B" of F i g u r e 8 i s c l a s s i f i e d a h y b r i d b a c k c r o s s because it p o s s e s s e s  results  p a t t e r n i n d i c a t e s the u n l i k e l i h o o d of i t b e i n g  F^ hybrids possessed  the z o n e s 1, 2, 3, 4, (5)  A s s u m i n g F £ h y b r i d s do n o t e x i s t i n n a t u r e ,  MDH  this i n d i v i d u a l m u s t  be a B CM. The  c l a s s i f i c a t i o n o f h y b r i d t y p e s b a s e d on the v a r i o u s  of m u s c l e p r o t e i n a n d M D H  patterns  ispresented  combinations  i n T a b l e VII.  Individual  MUSCLE ZONES  F i g u r e 8.  PROTEINS I 23  III!  MALIC ZONES  I  DEHYDROGENASE 2 3 4 (5)  F l e c t r o p h e r o g r a m s of m u s c l e p r o t e i n a n d m a l i c d e h y d r o g e n a s e p a t t e r n s f r o m t h r e e n a t u r a l h y b r i d s f r o m Stave Lake(1967 s a m p l e s ) .  38  T A B L E VII.  C l a s s i f i c a t i o n of h y b r i d types b a s e d on the combinations of m u s c l e p r o t e i n and m a l i c dehydrogenase p a t t e r n s . " F j h y b r i d " c l a s s i f i c a t i o n may include some h y b r i d backc r o s s e s to e i t h e r s p e c i e s because of a s s o r t m e n t f o r F h y b r i d p a t t e r n s of both p r o t e i n s s i m u l t a n e o u s l y . x  Hybrid Clas sification "Fi  Hybrids"  Muscle Protein Zones  M a l i c Dehydrogenase Zones  1,2,3  and  1,2,3,4,(5)  Hybrid backcrosses to M y l o c h e i l u s ( B C M s )  2, 3 3 3 3 1,2,3  and and and and and  any p a t t e r n 1, 2, 3, 4, (5) 1,2,3 3, 4, (5) 3  H y b r i d b a c k c r o s s e s to Richardsonius  1,2 1 1,2,3  and and and  any p a t t e r n 1,2,3,4,(5) 1, 3, (5)  39 h y b r i d s c a n n o t be i d e n t i f i e d w i t h c e r t a i n t y b e c a u s e h y b r i d  backcrosses  to e i t h e r s p e c i e s c a n a s s o r t f o r the F ^ h y b r i d m u s c l e p r o t e i n a n d pattern simultaneously.  In a l i k e m a n n e r , h y b r i d b a c k c r o s s e s  s p e c i e s c a n a s s o r t f o r the r e s p e c t i v e p a r e n t a l p r o t e i n s  MDH  to e i t h e r  simultaneously.  Seven individuals with 9 anal f i n rays p o s s e s s p a r e n t a l M y l o c h e i l u s protein and M D H Mylocheilus,  patterns.  S o m e o f t h e s e i n d i v i d u a l s c o u l d be p u r e  a s i n d i v i d u a l s w i t h 9 a n a l f i n r a y s a r e o c c a s i o n a l l y found,  even i n a l l o p a t r i c populations. be  muscle  slightly underestimated.  So, the n u m b e r of h y b r i d b a c k c r o s s e s  might  40 Results P r e d o r s a l / P r e p e l v i c Length R a t i o s (D/P) and L a t e r a l L i n e Scales of M y l o c h e i l u s and R i c h a r d s o n i u s f r o m Stave L a k e and f r o m A r e a s Outside the H y b r i d Zone :  Richardsonius  f r o m Alouette L a k e , the m a j o r drainage s y s t e m west  of Stave Lake, w e r e c o m p a r e d w i t h those f r o m Stave L a k e , the h y b r i d zone. In both c h a r a c t e r s examined, l a t e r a l l i n e s c a l e s and the D/P  r a t i o , no  s i g n i f i c a n t d i f f e r e n c e s a r e o b s e r v e d i n the m e a n s of the two groups (Figs. 9 and 10, Table VIII).  S i m i l a r l y , M y l o c h e i l u s f r o m V a n c o u v e r I s l a n d and  the Seechelt P e n i n s u l a show no s i g n i f i c a n t d i f f e r e n c e s f r o m those of the h y b r i d zone i n the m e a n s of these c h a r a c t e r s (Figs. 9 and 10, Table VIII). If the l o c i c o n t r o l l i n g the n u m b e r of l a t e r a l line s c a l e s and the  D/P  r a t i o can be c o n s i d e r e d r e p r e s e n t a t i v e of the two genomes, swamping between M y l o c h e i l u s and R i c h a r d s o n i u s does not appear i n p r o g r e s s . C o m p o s i t i o n of the N a t u r a l H y b r i d P o p u l a t i o n i n Stave L a k e On the b a s i s of a n a l f i n r a y s and two b i o c h e m i c a l c h a r a c t e r s , an e s t i m a t e was made of the v a r i o u s h y b r i d s p r e s e n t i n Stave Lake.  Hybrids,  i n g e n e r a l , c o m p o s e d 6. 1 % of the c o m b i n e d M y l o c h e i l u s - R i c h a r d s o n i u s gene p o o l i n 1967.  Of these h y b r i d s , p r e s u m e d F^s c o m p r i s e 76. 3%;  p r e s u m e d 1st generation h y b r i d b a c k c r o s s e s to M y l o c h e i l u s , 15.4%; and p r e s u m e d 2nd o r l a t e r g e n e r a t i o n h y b r i d b a c k c r o s s e s to M y l o c h e i l u s , 6. 6 % ( T a b l e s I X and X). P r e s u m e d 1st and 2nd or l a t e r g e n e r a t i o n h y b r i d b a c k c r o s s e s to -Richardsonius compose 1. 7 % and 0. 0% of a l l h y b r i d s , r e s p e c t i v e l y .  41  VANCOUVER SEECHELT.  F i g u r e '9,  ISLAND PENINSULA  T h e r a t i o of p r e d o r s a l a  n  d  ^.i£jj£i;^££:liliL  withia  u r e o e i v i c u •nglh of M y l o c h e i l u s o u t s i d e I he h y b r i d z o n e .  a  n  d  F i g u r e 10  L a t e r a l l i n e s c a l e d i s t r i b u t i o n of M y l o c h e i 1 us a n d R i c h a r d s o n i u s w i t h i n and outside-- the h y b r i d ; - o « e .  T A B L E VIII.  Species  Mylocheilus  C o m p a r i s o n of m o r p h o l o g i c a l c h a r a c t e r s of M y l o c h e i l u s and R i c h a r d s o n i u s f r o m w i t h i n and outside the h y b r i d zone.  L a t e r a l Line Scales s t X  Area  Vancouver Island Sechelt Peninsula  72.7  2  X  7.1  0.980  D/P R a t i o s s  0.0005  f  r  [  \ 0.43 Mylocheilus  Stave L a k e  72.9  7.6  Richardsonius  Alouette L a k e  60.3  11.2  Richardsonius  Stave L a k e  60.1  7.5  ** i n d i c a t e s s i g n i f i c a n t " t " v a l u e s ,  « = .05  r [ 0.51  t  2  0.984  0.0006  1.203  0.0021  1.208  0.0001  1.55  ,  {  0.98  T A B L E IX.  M u s c l e p r o t e i n a n d m a l i c d e h y d r o g e n a s e p a t t e r n s of t h o s e i n d i v i d u a l s of the M y l o c h e i l u s - R i c h a r d s o n i u s rays.  gene p o o l s w i t h <8  S a m p l e s a r e f r o m the h y b r i d z o n e a n d m a y  o r > 15 a n a l f i n  include pure  i n d i v i d u a l s of e i t h e r s p e c i e s o r 2nd a n d l a t e r g e n e r a t i o n h y b r i d backcrosses.  Muscle Proteins Zones =  Mylocheilus Richardsonius  3  2, 3  1,2, 3  1, 2  249  -  -  -  Malic Dehydrogenase 1  3,(5)  248 178  3, 4,(5)  1,2, 3  1,2,3,4,(5)  1,3,(5)  -  1  -  178  45  TABLE  X.  Composition  of the M y l o c h e i l u s - R i c h a r d s o n i u s  of S t a v e L a k e i n 1967.  Separation  gene c o m p l e x  of h y b r i d t y p e s i s b a s e d  on the i n t e r i t a n c e of a n a l f i n r a y s , a g e n e r a l m u s c l e p r o t e i n , and m a l i c dehydrogenase. w i t h <8  and  As  s h o w n i n T a b l e IX,  250  fish  178 f i s h w i t h > 15 a n a l r a y s w e r e e x a m i n e d to  e s t i m a t e the n u m b e r s of s e c o n d o r l a t e r g e n e r a t i o n h y b r i d backcrosses  to M y l o c h e i l u s and R i c h a r d s o n i u s  Estimated T y p e of I n d i v i d u a l  Numbers  "Pure" Mylocheilus "Pure" Richardsonius Presumed F Presumed  x  Hybrids  respectively.  Percent Percent  of t o t a l  1536.3  80.5  255.0  13.4  89.0  4.7  76.3  18.0  0.9  15.4  7.7  0.4  6.6  2.0  0.1  1.7  0.0  0.0  1 st g e n e r a t i o n  hybrid backcrosses  to  Mylocheilus P r e s u m e d 2nd  or later  generation hybrid backc r o s s e s to M y l o c h e i l u s P r e s u m e d 1st g e n e r a t i o n hybrid backcrosses  to  Richardsonius P r e s u m e d 2nd  or l a t e r  generation h y b r i d backc r o s s e s to  Richardsonius  of  a l l hybrids  0.0  46 T h e s e d a t a i n d i c a t e that F ^ h y b r i d s a r e q u i t e a b u n d a n t i n S t a v e while h y b r i d b a c k c r o s s individuals decrease b a c k c r o s s generation. tage. two  Consequently,  i n f r e q u e n c y b e y o n d the f i r s t  T h i s s u g g e s t s that t h e y a r e at a s e l e c t i v e s w a m p i n g i s p r e v e n t e d b e t w e e n the two  disadvan-  s p e c i e s ; the  gene p o o l s m a i n t a i n t h e i r g e n e t i c i n t e g r i t y . The  h y b r i d p o p u l a t i o n i n 1966  combined. or  Lake,  15  r e p r e s e n t s 5. 5%  of the two  gene p o o l s  H o w e v e r , this e s t i m a t e e x c l u d e s any h y b r i d s with 8 o r l e s s  or g r e a t e r a n a l f i n r a y s because an e l e c t r o p h o r e t i c a n a l y s i s  not p e r f o r m e d on t h e s e f i s h i n 1966.  was  If the a b o v e m e n t i o n e d h y b r i d s a r e .  s u b t r a c t e d f r o m the 1967  h y b r i d p o p u l a t i o n e s t i m a t e , the two y e a r s d a t a  a r e c o m p a r a b l e ; the 1967  h y b r i d p o p u l a t i o n i s then e s t i m a t e d a t 5. 7 % ,  e s t i m a t e v e r y c l o s e to that o b t a i n e d i n  i  1966.  an  47 Isolating M e c h a n i s m s In p r e v i o u s and  s e c t i o n s o f the t h e s i s i t w a s d e t e r m i n e d that M y l o c h e i l u s  Richardsonius  a r e reproductively isolated,  extensive hybridization.  e v e n i n the p r e s e n c e of  If r e p r o d u c t i v e i s o l a t i o n t r u l y e x i s t s ,  m e c h a n i s m s m u s t be o p e r a t i v e .  isolating  T h i s s e c t i o n of the t h e s i s a t t e m p t s to  d e s c r i b e what those m e c h a n i s m s a r e .  M a t e r i a l s and M e t h o d s Obse rvations Spawning populations of M y l o c h e i l u s , hybrids were observed of 1966 a n d 1967.  Richardsonius,  and  their  i n D e v i l s C r e e k d u r i n g the s p r i n g s a n d s u m m e r s  In 1966, o b s e r v a t i o n s w e r e m a d e t h r o u g h o u t the c r e e k ,  w h i l e i n 1967, they w e r e c o n c e n t r a t e d i n the l o w e r s e c t i o n .  T h i s section was  d i v i d e d into 20 m e t e r l e n g t h s w h e n the l a k e l e v e l w a s v e r y l o w (317 f e e t above sea level).  T h e 20 m e t e r s e c t i o n s b e g i n a t the l o w e r l o g j a m p o o l  and  terminate  The  l o w e r s e c t i o n w a s 325 m e t e r s l o n g at the b e g i n n i n g of o b s e r v a t i o n s  May  a t the p o i n t w h e r e D e v i l s C r e e k e n t e r s Stave L a k e ( F i g . 11).  1, 1967.  on  H o w e v e r , a s the l a k e l e v e l r i s e s the c r e e k i s i n n u n d a t e d  and d e c r e a s e s  i n length.  A n i n f r a - r e d v i e w e r was u s e d with l i m i t e d  success f o r nocturnal observations i n D e v i l s Creek.  W i t h the v i e w e r ,  the a d u l t s a p p e a r a s s i l v e r y o b j e c t s , the i d e n t i t y of w h i c h i s d i f f i c u l t to a s c e r t a i n e x c e p t f o r the l a r g e r type of M y l o c h e i l u s . ing behaviour  o f the two s p e c i e s a n d the h y b r i d s c o u l d o n l y be d e t e r m i n e d  in a crude fashion. and  A s a r e s u l t the s p a w n -  the h y b r i d s  In o r d e r to e s t i m a t e  the p r o p o r t i o n s of the two s p e c i e s  p r e s e n t i n the c r e e k d u r i n g a s p a w n i n g m i g r a t i o n , a 25  48  49 a n d / o r 38 m m  s t r e t c h m e s h g i l l n e t w a s p l a c e d a c r o s s the m o u t h a f t e r  the s p a w n i n g f i s h h a d e n t e r e d the c r e e k .  L a t e r , after spawning, l a r g e  n u m b e r s of f i s h w e r e c a p t u r e d a n d i d e n t i f i e d a s they r e t u r n e d to the l a k e . On some o c c a s i o n s spawning f i s h w e r e s u f f i c i e n t l y dense to p e r m i t sampling  b y hand.  Survival Experiments Egg Egg  S u r v i v a l to H a t c h i n g  s u r v i v a l e x p e r i m e n t s w e r e d e s i g n e d to t e s t the v i a b i l i t y o f e g g s  f r o m f e r t i l i z a t i o n to h a t c h i n g o f v a r i o u s c r o s s e s i n v o l v i n g M y l o c h e i l u s , Richardsonius,  and their hybrids.  Ripe m a l e s and females  were  collected  f r o m D e v i l s C r e e k f o r u s e i n the c r o s s i n g e x p e r i m e n t s .  M a l e s were  c o n s i d e r e d r i p e w h e n m i l t c o u l d be e x t r u d e d m a n u a l l y .  Transparency  and a d h e s i v e n e s s  o f the e g g s s e r v e d a s c i r t e r i a f o r r i p e f e m a l e s .  500 e g g s w e r e e x t r u d e d f r o m i n d i v i d u a l f e m a l e s dishes. the eggs.  100-  into w e t t e d p l a s t i c p e t r i  S e v e r a l d r o p s of m i l t w e r e e x t r u d e d f r o m a m a l e a n d m i x e d with T h e e g g s w e r e w a t e r h a r d e n e d f o r 15 m i n u t e s i n a s h a l l o w t r a y  containing D e v i l s C r e e k water.  T h e r e a f t e r , the e g g s w e r e  maintained  until hatching i n a Heath incubator s u p p l i e d with flowing D e v i l s C r e e k water. D e a d e g g s w e r e u s u a l l y r e m o v e d d a i l y f r o m e a c h l o t to p r e v e n t f u n g a l contamination  o f v i a b l e eggs.  T h e r i p e e g g s of a n i n d i v i d u a l f e m a l e w e r e  d i v i d e d into at l e a s t t h r e e l o t s a n d f e r t i l i z e d by the t h r e e t y p e s of m a l e s (Mylocheilus,  Richardsonius,  the v a r i a b i l i t y  caused  The for  a n d h y b r i d s ) . T h i s p r a c t i c e s e r v e s to c o n t r o l  by u s i n g u n r i p e f e m a l e s  i n the c r o s s e s .  p a r e n t s o f a l l c r o s s e s m a d e i n 1967, b u t n o t i n 1966, w e r e f r o z e n  subsequent electophoretic analysis.  50 S u r v i v a l of Y o l k - s a c  F r y to F i n g e r l i n g s  Y o l k - s a c f r y of M y l o c h e i l u s , R i c h a r d s o n i u s , r e c i p r o c a l F\ a n d p r e s u m e d F\ experimental  hybrid backcrosses  to M y l o c h e i l u s  hybrids,  were reared under  c o n d i t i o n s to d e t e r m i n e t h e i r r e l a t i v e v i a b i l i t i e s .  days after hatching,  Three  100 y o l k - s a c f r y f r o m e a c h of the a b o v e - m e n t i o n e d  c r o s s e s w e r e p l a c e d i n i d e n t i c a l p l y w o o d c o n t a i n e r s (50x50x50 c m ) they w e r e 63 d a y s o l d . c r o s s e s had  two  One  replicates.  l o t was  r e a r e d f o r o n l y 48 d a y s .  B e c a u s e of the h i g h m i d - s u m m e r  until  Most temperatures  of D e v i l s C r e e k , f l o w i n g w a t e r f r o m the c o l d e r c r e e k a d j a c e n t to i t s u p p l i e d w a t e r to e a c h r e a r i n g box.  A l l experimental  q u a n t i t i e s of i d e n t i c a l d i e t s of A r t e m i a L a k e , and f r o z e n adult b r i n e s h r i m p . ated e v e r y  15-20  days.  for future m o r p h o l o g i c a l  fry were maintained  nauplii, The  on  equal  l i v e plankton f r o m Stave  f r y f r o m each lot were e n u m e r -  A t the e n d of the e x p e r i m e n t a l l f i s h w e r e f r o z e n and b i o c h e m i c a l  examination.  51 Results P r e m a t i n g Isolating M e c h a n i s m s Seasonal Observations  on D e v i l s  Isolation  C r e e k were conducted f r o m M a y  A u g u s t 5 i n 1966 a n d f r o m M a y  1 to J u l y 11 i n 1967.  18 to  F i g u r e 12 g r a p h i c a l l y  i l l u s t r a t e s the t i m e I n t e r v a l d u r i n g w h i c h p o p u l a t i o n s of M y l o c h e i l u s , Richardsonius,  a n d t h e i r h y b r i d s (in s p a w n i n g c o n d i t i o n ) w e r e p r e s e n t i n  D e v i l s Creek.  In 1966, M y l o c h e i l u s a n d R i c h a r d s o n i u s  were already  p r e s e n t i n low n u m b e r s when o b s e r v a t i o n s w e r e begun on M a y were observed  t h r e e d a y s l a t e r o n M a y 21, 1966.  Although  18.  Hybrids  observations  w e r e b e g u n o n the f i r s t of M a y i n 1967, f i s h i n s p a w n i n g c o l o r a t i o n w e r e not o b s e r v e d  until M a y  17.  It w a s a p p a r e n t that q u a l i t a t i v e l y , a t l e a s t ,  the s p a w n i n g p e r i o d s o f the two s p e c i e s o v e r l a p to a g r e a t e x t e n t i n D e v i l s Creek.  T h e spawning p e r i o d of R i c h a r d s o n i u s  on a l a t e r date than M y l o c h e i l u s .  On a quantitative b a s i s , it was  that the p e a k o f s p a w n i n g f o r M y l o c h e i l u s 15 i n 1967 ( T a b l e s X I , XII).  i n 1966 a n d 1967 t e r m i n a t e d  w a s June 16 i n 1966 a n d June 13-  T h e spawning peak of R i c h a r d s o n i u s  d e t e r m i n e d to b e a w e e k o r so l a t e r .  estimated  However,  was  c o m p l e t e quantitative data  w e r e not c o l l e c t e d . H y b r i d f i s h e s a r e p r e s e n t i n D e v i l s C r e e k d u r i n g m o s t of the i n t e r v a l in which M y l o c h e i l u s ,  a n d d u r i n g p a r t o f the i n t e r v a l i n w h i c h  Richardsonius,  a r e p r e s e n t ( F i g . 12). T h e p e a k of h y b r i d a b u n d a n c e a p p e a r s to c o i n c i d e c l o s e r to the p e a k of M y l o c h e i l u s a b u n d a n c e ( T a b l e s X I , XII).  a b u n d a n c e than the p e a k o f R i c h a r d s o n i u s  1966 MYLOCHEILUS HYBRIDS RICHARDSONIUS  .  .  ...  ^^^^k-^kM^i^M^^^. _.  2 3 4 5 6 7 S 9 iO I! !2 13 14 15 16 17 i8 S3 20 2S 22 23 24 25 2S 27 28 23 30 3! MAY  ^ipSf  Pi  is |~| |  JUNE  1967  j HYBRIDS  . ^  j. _  I 23 45 6 7 8 9  111  HYBRIDS RICHARDSONIUS I g u r e .12.  6 7 8 9 10 I!  JULY  I MYLOCHEILUS  vlYLOCHEILUS  JUNE  p| - - p|-fevi— m-  6 7 8 9 iO Ii i2 13 14 15 16 17 !8 13 20 2! 22 23 24 25 2S 27 28 29 30 ! Z 3 4 5  RICHARDSONIUS  12 3 4 5  •  § M r  r  I:  ^  ^  ^  10'i! !2 13 14 (5 !6 IT 18 19 20 21 22 23 24.25 26 27 23 29 30 31 1 2 3 4 5 MAY JUNE  '  -  '1  t\"\  u * . ^ J U L « » 6 7 8 9 10 I! 12 13 14 15 (6 17 18 19 20 21 22 23 24 25 26 27 28 29 30 i 2 3 4 5 6 7 8 9 10 11 JUNE  JULY  Seasonal distribution of M y l o c h e i l u s , R i c h a r d s o n i u s , a n d t h e i r h y b r i d s i n D e v i l s C r e e k f o r 1966 a n d 1967. B l a c k e n e d a r e a s a r e days w h e n p a r t i c u l a r f i s h were: present. D a s h e d a r e a s a r e days when no o b s e r v a t i o n s w e r e m a d e .  TABLE  XI.  G i l l n e t c a t c h e s of M y l o c h e i l u s , R i c h a r d s o n i u s , d u r i n g the s p a w n i n g s e a s o n of 1966. hand.  and their h y b r i d s f r o m  * * I n c l u d e s one  G = s e x u a l l y m a t u r e f i s h , but n o t r i p e ;  R  Devils Creek  o b s e r v a t i o n of f i s h s a m p l e d  = sexually mature, ripe  by  fish;  Sp = s e x u a l l y m a t u r e , r i p e f i s h w h i c h h a v e d e p o s i t e d a l l o r m o s t of t h e i r g a m e t e s . R i c h a r d s o n i u s a r e not r e p r e s e n t e d i n t h e i r t r u e a b u n d a n c e b e c a u s e m a i n l y s t r e t c h m e s h gillnets w e r e used. Me Location Lower Jam  T h e s e a l l o w m o s t R i c h a r d s o n i u s to e s c a p e .  Mylocheilus  sh  dd  Size G  R  Hybrids  dd  ?? Sp  Date  mm  5-21  38  2  5-25 5-30  38  6 2  1  38  5-31  38  5  2  5-31 6-06  19 38  1  2  6-07  38  1  6-08  38  3 1  6-09 6-15 6-16  38  R  G  38mm  Sp  G  R  R i c h a r d s onius  dd  ?? Sp  G  R  Sp  G  R  ?$ Sp  G  R  Log  Pool  ." "  " " "  1 1  1  2  1  1  38 38  1 2 1  2  1  1 1  1  1  18  2  2  20  2  1 3  45  1  9  1  1 1  R i f f l e at 60' C r e e k length Lower Jam  6-16  1  2  1  2  Log  Pool  6-22  1  38 Totals  =  84  14  21  124  5  23  2 28  2  2  =  154  Sp  T A B L E XII. G i l l n e t c a t c h e s of M y l o c h e i l u s ,  Richardsonius,  d u r i n g the s p a w n i n g s e a s o n of 1967. R = sexually mature,  ripe fish;  and their hybrids  from Devils  Creek  G = s e x u a l l y m a t u r e f i s h , b u t not r i p e ;  Sp = s e x u a l l y m a t u r e ,  r i p e f i s h which have  deposited  a l l o r m o s t of t h e i r g a m e t e s . Mesh  Mylocheilus o"o-  . Size Location Lower Jam  Log  Pool  Devils Cr. Mouth Lower Jam  Log  Pool  Devils Cr. Mouth 11  Date  mm  5-17  38  9  5-19  25  3  5-26  38  9  6-02  38  15  6-02  38  6-06  25  11  6-07  25  r I  6-13  11  Devils Cr. Devils Cr. Mouth !t  G  R  R  Sp  G  R  Sp  G  R  do Sp  G  R  99 Sp  G  R  Sp  1  2  1  5  1  1 12  2  2  2  7  2  1  11  1  1 21  4  8  3  2  30  1  3  2  1  8  3  13  6  25  25, 38  65  6-14  25, 38  35  25, 38  9  6-16  25, 38  7  6-17  25  4  II  6-25  25  13  11  6-26  38  1  =  G  Richardsonius  99  8  6-15  Totals  Sp  Hybrids  9?  215  13 4  1  6  15 265  1  1  1  9 2  24 11  55  8  2  4  107  9 38 41  8  1 193  60 49  1  1  1 6  2  1  71  '  303  =  639  55 Spatial and T e m p o r a l (Diel) Isolation An and  attempt was m a d e to d e t e r m i n e if M y l o c h e i l u s ,  their hybrids a r e spatially and o r temporally  d u r i n g spawning.  E a c h evening f r o m  June 4  Richardsonius,  (dielly)  segregated  to June .17, 1967, the  d i s t r i b u t i o n of s p a w n i n g f i s h f r o m the l o w e r s e c t i o n o f D e v i l s C r e e k w a s recorded.  T h e data a r e g r a p h i c a l l y d e p i c t e d i n F i g u r e  13.  This figure  - i l l u s t r a t e s the l e n g t h o f D e v i l s C r e e k a n d the l o c a t i o n o f s p a w n i n g f i s h i n the c r e e k o n a g i v e n day. species occurs level.  c r e e k length,  riffles  the s p a w n i n g o f the two  on the f i r s t r i f f l e a b o v e the l a k e , r e g a r d l e s s of the l a k e  A s the l a k e l e v e l r i s e s d u r i n g M a y ,  decreasing ingly,  With few exceptions,  June, a n d J u l y , t h e r e f o r e ,  spawning a c c o r d i n g l y m o v e s upstream.  Interest-  o n w h i c h s p a w n i n g o c c u r r e d d u r i n g h i g h l a k e l e v e l s i n 1966,  w e r e n o t u s e d i n 1967 w h e n the l a k e l e v e l w a s l o w e r . Both s p e c i e s of f i s h enter D e v i l s C r e e k at a p p r o x i m a t e l y t i m e e a c h day.  Richardsonius  o n the a v e r a g e m a y e n t e r s l i g h t l y e a r l i e r  i n the day, b u t i t i s d i f f i c u l t to e s t i m a t e  accurately.  F i s h begin entering  D e v i l s C r e e k a s e a r l y a s 2130 h o u r s ( P a c i f i c D a y l i g h t S a v i n g a l t h o u g h o n s o m e d a y s they do n o t do so u n t i l 2245 h o u r s . w e r e o b s e r v e d o n the f i r s t  r i f f l e a s l a t e a s 0215 h o u r s .  r e t u r n e d to the l a k e b y d a y l i g h t . is v e r y high,  the s a m e  In those  Time),  Spawning f i s h  M o s t f i s h have  s i t u a t i o n s w h e r e the l a k e l e v e l  some f i s h move u p s t r e a m after spawning and seek.shelter i n  the d e e p p o o l a t the l o w e r l o g j a m i n D e v i l s C r e e k (See F i g . 11). A l t h o u g h i t i s quite a p p a r e n t that M y l o c h e i l u s a n d R i c h a r d s o n i u s  are  s p a w n i n g on the s a m e r i f f l e a t the s a m e t i m e , i n f o r m a t i o n o n the b e h a v i o u r  -200  JUNE  • JULY  DATE F i g u r e 13.  R e l a t i o n s h i p b e t w e e n l o c a t i o n of s p a w n i n g f i s h a n d the l e n g t h of D e v i l s C r e e k f o r p a r t i c u l a r d a t e s i n 1967. Crosses indicate c r e e k l o c a t i o n s of spawning fish.  57 of the h y b r i d s w a s d i f f i c u l t to o b t a i n s i n c e h y b r i d s a r e d i f f i c u l t to r e c o g n i z e i n the c r e e k .  H o w e v e r , two k i n d s of e v i d e n c e  i n fact, a r e not t e m p o r a l l y o r s p a t i a l l y s p e c i e s d u r i n g spawning.  First,  segregated  suggest  that h y b r i d s ,  f r o m the p a r e n t a l  g i l l n e t s s e t b e l o w the r i f f l e o n w h i c h  f i s h a r e spawning y i e l d s u b s t a n t i a l n u m b e r  s o f h y b r i d f i s h e s ( T a b l e XII).  S i n c e s p a w n i n g f i s h a r e e v i d e n t ( w i t h i n f r a - r e d v i e w e r ) o n l y o n the f i r s t r i f f l e a b o v e the l a k e d u r i n g a n y g i v e n e v e n i n g ,  hybrid fishes a r e likely  s p a w n i n g o n the s a m e r i f f l e a t the s a m e t i m e a s a r e M y l o c h e i l u s Richardsonius.  and  S e c o n d , a s a m p l e of s p a w n i n g f i s h c o l l e c t e d b y h a n d  f r o m a riffle area  18" s q u a r e o n June 16, 1966, r e v e a l e d that 24. 6 %  of t h e m  w e r e h y b r i d s ( T a b l e XIII ). A s w i t h the h y b r i d s c o l l e c t e d f r o m the p e l a g i c a r e a o f the l a k e ,  those  h y b r i d s i n s p a w n i n g c o l o r a t i o n f r o m D e v i l s C r e e k a p p e a r to be p r i m a r i l y of a F j n a t u r e .  D a t a o n the a n a l f i n r a y d i s t r i b u t i o n of h y b r i d s  f r o m D e v i l s C r e e k i n 1967 a r e p r e s e n t e d  below;  Anal F i n Rays Date  9  June 7  10  11  3  10  13  1  2  1  15  1  5  8 2  16 17 25  12_  1  2  4  5  8  26  3 3  17 •  36  0  collected  58  T A B L E XIII.  Composition  of a s a m p l e of the s p a w n i n g p o p u l a t i o n f r o m  D e v i l s C r e e k at 2245 h o u r s P.D.T., J u n e 16, 1966. f i s h were collected by hand f r o m  an  Spawning  18" s q u a r e r i f f l e  area  at the 20 m e t e r c r e e k l e n g t h ( s e e F i g u r e 11).  Percentage Female s  Males  Total  Total  11  45  56  72.7  R i c h a r d s onius  0  2  2  2.7  Hybrid  1  18  19  24.6  12  65  77  Mylocheilus  Totals =  59 H y b r i d s w i t h 9 a n a l f i n r a y s , thought on the b a s i s of e x p e r i m e n t a l (Fig.  7) to be p r i m a r i l y h y b r i d b a c k c r o s s e s  O n l y 3 out of 5.6 h y b r i d s p o s s e s s e d  such a number.  w i t h 11 a n a l f i n r a y s a r e v e r y abundant, are  to M y l o c h e i l u s  ,are  crosses scarce.  In c o n t r a s t ,  those  s u g g e s t i n g that m o s t of the h y b r i d s  FjS. Ethological Isolation I n f o r m a t i o n on the s p a w n i n g b e h a v i o u r  and  t h e i r h y b r i d s was  of M y l o c h e i l u s ,  Richardsonius  d i f f i c u l t to o b t a i n b e c a u s e s p a w n i n g o c c u r s on r i f f l e s  d u r i n g h o u r s of d a r k e n s s .  Generally,  i t was  d e t e r n i n e d that b o t h  a r e m a s s spawners with m a n y m a l e s attending a single female. X I a n d XII r e f l e c t the g r e a t a b u n d a n c e of m a l e s o v e r f e m a l e s .  species  Tables Whether  F ^ h y b r i d s a r e p r o d u c e d a s the r e s u l t of m i s - m a t i n g s b e t w e e n the s p e c i e s o r a s the r e s u l t of c h a n c e m e e t i n g of g a m e t e s of two mated species'groups of h y b r i d s was 23. 8 %  was  not d e t e r m i n e d .  not o b s e r v e d  properly  spawning  behaviour  but s o m e i n f e r e n c e s c a n be m a d e .  First,  of a l l h y b r i d f e m a l e s c a p t u r e d i n D e v i l s C r e e k w e r e s p e n t  to 15. 6 %  for Mylocheilus females,  t h e i r ripe eggs.  T h e r e i s also evidence  h u n d r e d e g g s s p a w n e d on the e v e n i n g r i f f l e at the 20 m  compared  s u g g e s t i n g that h y b r i d f e m a l e s do d e p o s i t that h y b r i d e g g s a r e f e r t i l i z e d  a n d a r e v i a b l e a t l e a s t to the " e y e d " stage.  On  June 20,  1966,  several  of J u n e 16 w e r e c o l l e c t e d f r o m the  c r e e k length location.  l a b o r a t o r y u n t i l the r e s u l t i n g f r y h a d The  Likewise,  two  T h e s e e g g s w e r e r e a r e d i n the  their anal fin rays fixed in number.  d i s t r i b u t i o n of a n a l f i n r a y s f r o m t h e s e f i s h i s s h o w n i n F i g u r e  T h o s e f i s h w i t h "9" a n a l f i n r a y s p r e s u m a b l y r e p r e s e n t h y b r i d  14.  backcrosses  (195)  N - 325  b  -^  i n;  \  1S\  W5« :  9  e 14.  10  12  ANAL FIN  13  i  14  *  i f°  1 17.  18  19  20  RAYS - NUMBERS  A n a l fin. r a y d i s t r i b u t i o n o f f i n g e r l i n g s r e a r e d f r o m e g g s c o l l e c t e d on J u n e 2 0 , 1966, f r o m a n 18" s q u a r e s e c t i o n o f the ' " 2 0 m e t e r " r i f f l of D e v i l s C r e e k .  61  to M y l o c h e i l u s sterile,  (See F i g . 7).  S i n c e h y b r i d m a l e s a r e , f o r the m o s t p a r t  (to be d i s c u s s e d s u b s e q u e n t l y ) t h e s e b a c k c r o s s e s a r e l i k e l y the  r e s u l t of h y b r i d 5$ x M y l o c h e i l u s dd correct,  some h y b r i d females,  crosses.  If t h e s e i n f e r e n c e s a r e  a t l e a s t , a r e a b l e to c o m p l e t e  spawning  successfully. The  r o l e that h y b r i d m a l e s p l a y i n the s p a w n i n g a g g r e g a t i o n s i s  unknown. Postmating Egg  Isolating M e c h a n i s m s  S u r v i v a l to H a t c h i n g  ' H y b r i d s t e r i l i t y a n d i n v i a b i l i t y a r e c o n s i d e r e d to be i m p o r t a n t p o s t mating  isolating m e c h a n i s m s in many animal species.  t h e y w e r e s t u d i e d i n the two  Consequently,  s p e c i e s u n d e r d i s c u s s i o n to see what i m p o r -  t a n c e c a n be a t t r i b u t e d to t h e m . made utilizing all combinations  Eighty-two  experimental crosses were  of M y l o c h e i l u s , R i c h a r d s o n i u s ,  p r e s u m e d F i h y b r i d m a l e s and females. a 3 x 3 f a c t o r i a l design (Winer,  1962  ) was  A n a n a l y s i s of v a r i a n c e f o r u s e d to t e s t f o r s i g n i f i c a n t  d i f f e r e n c e s i n m e a n s u r v i v a l between m a l e s and females.  The  s i g n i f i c a n c e i n t h e s e t e s t s , as i n a l l o t h e r s i n t h i s t h e s i s , w a s C l e a r l y , m a l e and f e m a l e effects are significant 35. 19 f o r f e m a l e s >  F a  _ = . 05, c  v a l u e s f o r m a i n effects ( m a l e ,  - , - 1'5). df = 2,73 =3  0  0  and  l e v e l of . 05.  ( F = 114. 27 f o r m a l e s ,  In v i e w of s i g n i f i c a n t  f e m a l e ) , a N e w m a n - K e u l s t e s t was  test f o r significant d i f f e r e n c e s between individual c e l l m e a n s in an sequence (Winer,  1962). T h e  them are given in Table  XIV.  "F"  to  u s e d to ordered  c e l l m e a n s and tests of s i g n i f i c a n c e between  62  TABLE  XIV.  M e a n e g g s u r v i v a l of e x p e r i m e n t a l  c r o s s e s to hatching and  t e s t s of s i g n i f i c a n c e b e t w e e n o r d e r e d p a i r s of m e a n s u s i n g the N e w m a n - K e u l s p r o c e d u r e ( W i n e r , and  1962).  1966, 1967  1968 d a t a c o m b i n e d .  M y l o c h e i l u s ?$  Cell Means Hybrid??  Richardsonius??  M y l o c h e i l u s o " 0"  71.6  65.4  91.5  Hybrido'cr  15.9  11.5  44.6  R i c h a r d s o n i u s 0* 0"  56.0  64.4  88.0  N e w m a n - K e u l s T e s t of O r d e r e d M e a n s Mean  Difference  Between Ordered  Pairs  Ordered  Number  of S t e p s  Between  Pairs  Ordered  Pairs  C r i t i c a l V a l u e s oi Sxq.95(r,73)  55.7** 40.1**  3  32.6  2  27.1  M?Hc? a n d R?Ho"  28.7**  2  27.1  R ? R d ' a n d R?Ho"  43.4**  2  27.1 32.6  H?Ho* a n d R?Ho*  46.9** 52.9** 33.1**  3 2 3  32.6  H?Hc? a n d H?Mo-  53.9**  3  32.6  M ? M c ? a n d M?Ro"  15.6  2  27.1  M?Mo* a n d R?Mo"  19.9 6.2  2  27.1  2  27.1  8.4  2  27.1  R?Rd' and R?Mc?  3.5  2  27.1  R?Ro" a n d M ? R d "  32.0  3  32.6  R?Ro" a n d H ? R c f  M?HcT a n d M?Mc? M?Ho" a n d M?Ro*  R 9 M 0 * a n d R?Ho" H?Hc? a n d H?Ro"  M?Mo" and H?Mc? M ? R r f a n d H$Ro*  27.1  23.6  2  H?Mo* a n d H?Ro"  1.0  2  27.1 27.1  H$Mo" a n d R?Mo"  26.1  '3  32.6  H?Ho" a n d M?Ho"  4.4  2 •  27.1  M"  = Mylocheilus,  .** - s i g n i f i c a n t  "R" = R i c h a r d s o n i u s ,  ,  and "H" = p r e s u m e d F  T  Hybrid  63 Crosses  of M y l o c h e i l u s  9 9 x R i c h a r d s o n i u s d* d" a n d the r e c i p r o c a l do  not d i f f e r s i g n i f i c a n t l y i n m e a n e g g s u r v i v a l f r o m p u r e c r o s s e s of e i t h e r parental species. Backcrosses  of p r e s u m e d  h y b r i d s to M y l o c h e i l u s  g i v e two s i g n i -  ficantly different r e s u l t s depending on w h i c h r e c i p r o c a l i s used. h y b r i d dd x M y l o c h e i r u s $ ? c r o s s e s p r o d u c e  a m e a n s u r v i v a l o f 15. 9 % a s  c o m p a r e d to 71. 6 % f o r p a r e n t a l M y l o c h e i l u s difference.  c r o s s e s - - a significant  F l hybrid 99 x Mylocheilus  The reciprocal crosses,  F^  dd,  y i e l d a m e a n s u r v i v a l o f 65. 4 % , a m e a n s u r v i v a l w h i c h i s n o t d i f f e r e n t from parental Mylocheilus Similarly,  backcrosses  crosses  (71.6%).  of p r e s u m e d F ^ h y b r i d s to R i c h a r d s o n i u s  y i e l d different r e s u l t s depending upon w h i c h r e c i p r o c a l i s used. c r o s s e s of p r e s u m e d F^ h y b r i d  9 9 x Richardsonius  different f r o m parental Richardsonius involving poorer  Richardsonius  crosses.  0*0*  Back-  a r e not s i g n i f i c a n t l y  However,  backcrosses  $$ a n d p r e s u m e d Fj_ h y b r i d dd y i e l d s i g n i f i c a n t l y  s u r v i v a l than p a r e n t a l R i c h a r d s o n i u s  crosses.  P r e s u m e d F ^ h y b r i d s w h e n c r o s s m a t e d p r o d u c e the p o o r e s t s u r v i v a l o f a n y c r o s s , o n l y 11. 5 % . n o t i n s i g n i f i c a n t , the a b n o r m a l i t y  Although  mean  a n e g g s u r v i v a l o f 11. 5 % i s  of n e w l y hatched  F £ fry m a k e s it unlikely  that they e x i s t i n n a t u r e . Of a l l c r o s s e s made, those w h i c h d i f f e r s i g n i f i c a n t l y a l w a y s one  thing i n c o m m o n ;  possess  at l e a s t one o f the c r o s s e s h a s a p r e s u m e d F T  h y b r i d a s the m a l e p a r e n t .  H o w e v e r , w i t h i n those c r o s s e s u t i l i z i n g  p r e s u m e d F ^ h y b r i d males, better egg s u r v i v a l is obtained with  Richardsonius  64 t h a n w i t h p r e s u m e d F± h y b r i d o r M y l o c h e i l u s  females.  This  fact  f u r n i s h e s a c l u e a s to the c a u s e of the p o o r e g g s u r v i v a l of the a b o v e crosses.  If'F^ h y b r i d m a l e s a r e e x t r e m e l y sterile, they should not have  b e e n a b l e to f e r t i l i z e the e g g s of R i c h a r d s o n i u s  females.  B u t they do. 99 x  T h i s s u g g e s t s that a t l e a s t p a r t of the m o r t a l i t y of M y l o c h e i l u s  p r e s u m e d F v h y b r i d dd a n d p r e s u m e d F ^ h y b r i d $ 9 x p r e s u m e d F ^ h y b r i d dd c r o s s e s m a y b e a t t r i b u t e d to g e n e t i c i n c o m p a t a b i l i t y o r i n v i a bility. However,  t h e r e i s s o m e e v i d e n c e that p r e s u m e d F ^ h y b r i d  undergo a b n o r m a l spermatogenesis and gonadal development, them partially sterile.  males  leaving  T h e testes of these m a l e s a r e c o n s i d e r a b l y  s m a l l e r than those of e i t h e r p a r e n t a l s p e c i e s .  T h e q u a n t i t y of m i l t w h i c h c a n  c a n be e x t r u d e d i s m a r k e d l y r e d u c e d , a n d i t s t r a n s p a r e n c y i s g r e a t , i n c o n t r a s t to that f r o m M y l o c h e i l u s  or Richardsonius.  A l t h o u g h the  q u a n t i t y o f s p e r m i n the m i l t d o e s n o t a p p e a r r e d u c e d i n p r e s u m e d F ^ hybrids  c o m p a r e d to e i t h e r s p e c i e s ,  a microscopic  e x a m i n a t i o n o f the  m i l t r e v e a l s that the F-^ h y b r i d s p e r m i s m o r e v a r i a b l e i n s i z e . T h e r e i s a l s o s o m e e v i d e n c e that the f a c t o r ( s ) i n the F ^ h y b r i d m a l e p r o d u c i n g p o o r e g g s u r v i v a l o f M 0 9 x Hdd  a n d H 9 9 x Hdd  crosses  a t e d i n a t l e a s t s o m e o f the h y b r i d b a c k c r o s s to M y l o c h e i l u s  is elimin-  males.  In  o t h e r w o r d s , i t s e e m s that a s s o r t m e n t f o r the f e r t i l i t y a n d v i a b i l i t y f a c t o r s of M y l o c h e i l u s  has o c c u r r e d .  T h e eggs of a single M y l o c h e i l u s  w e r e split into five lots and f e r t i l i z e d w i t h d i f f e r e n t m a l e s . s u r v i v a l was as follows:  female  T h e egg  M236  The  # Eggs Surviving  % Survival  1,695  1,379  81.4  R 241  640  413  64. 5  R 242  878  767  87.3  M239  "  "M"  Total Eggs  "  H240  942  16  1. 7  "  H238  871  803  92. 2  = Mylocheilus,  "R"  = Richardsonius,  "H"  =  hybrid.  d a t a i l l u s t r a t e the g r e a t d i s p a r i t y b e t w e e n h y b r i d m a l e s .  With hybrid m a l  m a l e "240", e g g s u r v i v a l i s o n l y 1. 7 % , b u t i s 92. 2 % w i t h h y b r i d m a l e "238." The  muscle protein and M D H  p a t t e r n s o f h y b r i d m a l e " 2 3 8 " r e v e a l that i t  is at least a first generation hybrid backcross  to M y l o c h e i l u s .  F o r hybrid  m a l e "240", the p r o t e i n p a t t e r n s a r e o f a F± h y b r i d n a t u r e .  S u r v i v a l of Y o l k - s a c The  F r y to F i n g e r l i n g s  s u r v i v a l o f y o l k - s a c f r y to the f i n g e r l i n g s t a g e w a s e x a m i n e d  under experimental  c o n d i t i o n s to d i s c o v e r w h e t h e r h y b r i d i n d i v i d u a l s a r e  i n v i a b l e r e l a t i v e to the p a r e n t a l s p e c i e s .  S u c h i n v i a b i l i t y c a n be a p a r t i a l  or complete postmating isolating mechanism.  T w o - w a y a n a l y s e s of  v a r i a n c e w e r e u s e d to t e s t f o r d i f f e r e n c e s i n s u r v i v a l b e t w e e n c r o s s e s (Winer,  1962).  not d i f f e r  S u r v i v a l of M y l o c h e i l u s  significantly f r o m parental Mylocheilus = 18  a  9 9 x Richardsonius  = . 05, d f = 1, 2  dd c r o s s e s do  c r o s s e s ( F =.001< F  5) '  '  Similarly,  Richardsonius  g$ x M y l o c h e i l u s  s u r v i v a l does not deviate significantly f r o m p a r e n t a l M y l o c h e i l u s (F=  0.57< F ^  „  0*0*  crosses  ^ ,, 18,5). H o w e v e r , a f t e r 48 d a y s of r e a r i n g , s u r =  v i v a l of h y b r i d b a c k c r o s s e s  to M y l o c h e i l u s p r o v e s to be s i g n i f i c a n t l y l o w e r  than p a r e n t a l M y l o c h e i l u s c r o s s e s by a p p r o x i m a t e l y  2 0 % (F•= 21.43>  F a  -~. 0 b j  66 —  - Mn^CHEILUS* m O C H E l L U S • MYLOCHEILUS? x.RICHARDSONiUS<f RlCHARDSGlilUS x RjCHARDSOMUS  * ^ I t P C H E J L U S x MYLOCJlE|iJJS  0  4  8  12  16 2 0 2 4 2 8 3 2 3 6 4 0 4 4 4 8 5 2 5 6 6 0 6 4  TIME F i g u r e 15.  - DAYS  S u r v i v a l o f y o l k - s a c f r y to the f i n g e r l i n g s t a g e u n d e r experimental conditions.  df = 1,13=  4 6 7  >  ( F i  ^ >15  M  O n l y one l o t of p a r e n t a l R i c h a r d s o n i u s  f r y was  r e a r e d b e c a u s e of  the d i f f i c u l t y of p r o c u r i n g r i p e f e m a l e s w h e n the e x p e r i m e n t s w e r e s t a r t e d . ' For  an  unknown reason,  this lot has  the p o o r e s t s u r v i v a l of any t e s t e d  . ( F i g . 15). An  i n s p e c t i o n of the d a t a r e v e a l s that a c o n s i d e r a b l e p r o p o r t i o n of  the t o t a l m o r t a l i t y (with the e x c e p t i o n of the p a r e n t a l R i c h a r d s o n i u s  cross)  i s u s u a l l y e n c o u n t e r e d b y the f i r s t e n u m e r a t i o n p e r i o d (16 o r 19 d a y s ) . The  r a t e of m o r t a l i t y s u b s e q u e n t to t h i s i s s u b s t a n t i a l l y l o w e r .  the f i r s t i n t e r v a l , food source may In s h a r p  the s w i t c h - o v e r  f r o m a n e n d o g e n o u s to a n e x o g e n o u s  h a v e a c c o u n t e d f o r the c o n s i d e r a b l e m o r t a l i t y  c o n t r a s t , l i t t l e m o r t a l i t y was  parental' R i c h a r d s o n i u s  cross.  tality increased, producing  Fj  hybrids.  The  In s u m m a r y ,  experienced.  s u f f e r e d the f i r s t 19 d a y s i n the  S u b s e q u e n t l y , h o w e v e r , the r a t e of m o r -  the p o o r e s t s u r v i v a l of a n y lot.  the v e r y p o o r s u r v i v a l of the R i c h a r d s o n i u s check Richardsonius  During  It i s f e l t that  lot is an anomaly.  f r y w e r e r e a r e d i n the l a b o r a t o r y i n 1968  s u r v i v a l of b o t h w a s  As  a  along  with  good and n e a r equal.  the v i a b i l i t y of p r e s u m e d F-^ h y b r i d  backcrosses  to  r  Mylocheilus  is markedly poorer  than M y l o c h e i l u s  c r o s s e s or for either  r e c i p r o c a l F-^ h y b r i d c r o s s e s . A b n o r m a l i t i e s of E x p e r i m e n t a l S o m e f i s h , u s e d i n the e x p e r i m e n t a l  Animals  s u r v i v a l s t u d i e s but not u s e d f o r  b i o c h e m i c a l s t u d i e s , w e r e e x a m i n e d f o r o b v i o u s a b n o r m a l i t i e s at the t e r m i n a t i o n of the e x p e r i m e n t .  The  P r o g e n y of p a r e n t a l M y l o c h e i l u s  results are presented and  reciprocal F  1  in Table  crosses are  XV. almost  68  T A B L E XV.  G r o s s a b n o r m a l i t i e s of f i s h u s e d  i n the v i a b i l i t y e x p e r i m e n t s  f r o m the y o l k - s a c f r y t o the f i n g e r l i n g  Number Female  Male  Examined  stage.  Number Abnormal  Mylocheilus  Mylocheilus  13  0  Mylocheilus  Mylocheilus  112  0  Mylocheilus  R i c h a r d s onius  88  1  Mylocheilus  Richardsonius  2  0  Richardsonius  Mylocheilus  75  0  Mylocheilus  "Hybrid"  30  0  "Hybrid"  Mylocheilus**  64  6  T y p e of A b n o r m ality  3 fish very  small,  3 f i s h with bulging abdomens "Hybrid"  Mylocheilus  37  2 f i s h with bulging abdomens 2 f i s h with light p i g m e n t a t i on 1 f i s h with light pigmentation  and  bulging abdomen 1 f i s h with d e f o r m e d vertebral ** some contamination  of M $  X Ho" c r o s s e s  column  69 f r e e of g r o s s a b n o r m a l i t i e s . to M y l o c h e i l u s  In c o n t r a s t , p r o g e n y of h y b r i d b a c k c r o s s e s  p o s s e s s many.  F o r t h r e e s u c h c r o s s e s , 1 1 . 5 % of the  i n d i v i d u a l s p o s s e s s e d distended abdomens, u n u s u a l l y light o r d a r k body pigmentation,  o r deformed vertebral columns(Fig.  16). A l t h o u g h  f i s h a r e n o t c o n s i d e r e d a s m o r t a l i t i e s i n the f r y s u r v i v a l  deformed  experiments,  it i s p o s s i b l e that s u c h i n d i v i d u a l s w o u l d be s e l e c t i v e l y e l i m i n a t e d u n d e r natural conditions.  Evidence  that t h i s o c c u r s c o u l d h a v e b e e n  i f l a r g e n u m b e r s o f a d u l t h y b r i d b a c k c r o s s e s to M y l o c h e i l u s  determined  had been  obtained, f o r they should c o n t a i n m a n y d e f o r m e d i n d i v i d u a l s i f s e l e c t i o n h a s riot o p e r a t e d a g a i n s t them. H y b r i d b a c k c r o s s e s to R i c h a r d s o n i u s w e r e n o t c o n s i d e r e d b e c a u s e of a s h o r t a g e of e x p e r i m e n t a l a n i m a l s .  Sex One  r a t i o s of h y b r i d a n d p a r e n t a l s p e c i e s ' populations  h u n d r e d a n d nine h y b r i d i n d i v i d u a l s s a m p l e d f r o m Stave L a k e d u r i n  the s u m m e r of 1967 w e r e e x a m i n e d t o d e t e c t a n y d e v i a t i o n s f r o m a 1: 1 sex ratio w h i c h m a y s e r v e as a postmating i s o l a t i n g m e c h a n i s m . b a s i s of b i o c h e m i c a l c h a r a c t e r i s t i c s w e r e d i v i d e d i n t o two g r o u p s , c r o s s e s to M y l o c h e i l u s . captured f r o m  (muscle proteins, MDH),  O n the the h y b r i d s  1) p r e s u m e d F ^ h y b r i d s a n d 2) h y b r i d b a c k -  T h e s e x r a t i o s o f the p a r e n t a l s p e c i e s ' f o r m s  Stave L a k e ( 1 " g i l l n e t s ) w e r e a l s o e x a m i n e d a n d a r e s h o w n  b e l o w w i t h the s e x r a t i o s of h y b r i d i n d i v i d u a l s .  70  A. D E F O R M E D  VERTEBRAL  COLUMN  B. N O R M A L C. D I S T E N D E D  ABDOMEN  D. N O R M A L  E. A B N O R M A L  F i g u r e 16.  PIGMENTATION  T y p i c a l a b n o r m a l i t i e s of p r o g e n y f r o m h y b r i d to M y l o c h e i l u s .  backcrosses  71 Mylocheilus  Richardsonius  Presumed  Hybrid  Backcrosses  F j Hybrids  To Mylocheilus  Males  279  24  43  10  Females  212  100  46  8  Sex  1:0. 76  Ratio  1:4.  17  1:1.  1:0.  07  80  (cTcf : $?) Chi-Square  9. 14*  * = s i g n i f i c a n c e at  a  46. 58*  0. 10  0. 22  = 0. 05  S u r p r i s i n g l y , b o t h p a r e n t a l f o r m s d e v i a t e s i g n i f i c a n t l y f r o m a 1:1 r a t i o , but i n d i f f e r e n t w a y s .  For Mylocheilus,  sex  there are m o r e m a l e s  than' f e m a l e s w h i l e the r e v e r s e is t r u e f o r R i c h a r d s o n i u s .  Why  these  s e x r a t i o s a r e f o u n d i s u n k n o w n , but p o s s i b l e f a c t o r s r e s p o n s i b l e a r e ; 1)  a n i n t r i n s i c a l l y a b n o r m a l sex r a t i o ,  two  s e x e s s u b s e q u e n t to s p a w n i n g , a n d  2) d i f f e _ r e n t i a l m o r t a l i t y of the 3) g i l l n e t s e l e c t i v i t y .  The  sex  r a t i o s of s p a w n i n g i n d i v i d u a l s of b o t h s p e c i e s i n D e v i l s C r e e k h e a v i l y favor males. P r e s u m e d F^ h y b r i d s and h y b r i d b a c k c r o s s e s S t a v e Lake^ ( 1" g i l l n e t s ) cantly different f r o m  1:1.  both p o s s e s s Although  to M y l o c h e i l u s  s e x r a t i o s w h i c h a r e not  from signifi-  the h y b r i d d a t a a r e d i f f i c u l t to i n t e r -  p r e t i n v i e w of the s e x r a t i o s of the p a r e n t a l s p e c i e s , i t i s p r o b a b l y reasonable  to c o n c l u d e  that the s e x r a t i o of h y b r i d s i s not a  i s o l a t i n g m e c h a n i s m b e t w e e n the two  C o m p a r i s o n of F i n g e r l i n g a n d The  postmating  species.  Adult H y b r i d  F i n Ray D i s t r i b u t i o n s  a n a l f i n r a y d i s t r i b u t i o n s of f i n g e r l i n g a n d adtilt h y b r i d s w e r e  c o m p a r e d to o b t a i n a r o u g h e s t i m a t e  of h y b r i d v i g o r o r h y b r i d i n v i a b i l i t y .  B a s e d o n the k n o w l e d g e of the h e r i t a b i l i t y of a n a l f i n r a y s , those  hybrids  w i t h 9 a n a l f i n r a y s a r e p r e s u m e d to be h y b r i d b a c k c r o s s e s  to M y l o c h e i l u s ,  w h i l e t h o s e w i t h 11 a n a l r a y s a r e p r e s u m e d to be p r i m a r i l y  hybrids.  If F\ h y b r i d s a n d h y b r i d b a c k c r o s s e s  to M y l o c h e i l u s  t i v e to the p a r e n t a l s p e c i e s , t h e i r f r e q u e n c y they b e c o m e .  C o n s e q u e n t l y , the f r e q u e n c y  a n a l f i n r a y s was  should decrease  rela-  the o l d e r  of t h o s e h y b r i d s w i t h 9 a n d 11  c o m p a r e d r e l a t i v e to M y l o c h e i l u s  a s a d u l t s ( F i g . 17).  are inferior,  as f i n g e r l i n g s  and  T h e h y b r i d s w o u l d h a v e b e e n c o m p a r e d to b o t h  p a r e n t a l s p e c i e s , but it was b e c a u s e of t h e i r s m a l l s i z e .  d i f f i c u l t to c a p t u r e f i n g e r l i n g  Richardsonius  T h e data a r e a s f o l l o w s :  Hybrids  Hybrids  Number  "With 9  W i t h 11  of  Anal Rays  Anal Rays  Mylocheilus  R a t i o of the R a t i o of the N u m b e r of N u m b e r of Hybrids  Hybrids  With 9 Anal Rays  W i t h 11 Anal Rays  to Mylocheilus  to Mylocheilus  18  647  0.0108:1  0. 0278:1  19  64  1,544  0.0123:1  0. 0415:1  1967 F i n g e r - 137  79  2* 258  0.0607:1  0. 0350: 1  1966 A d u l t s  7  1967 A d u l t s  ^  lings  If i t c a n be a s s u m e d that 1967 f i n g e r l i n g r e c r u i t m e n t w a s quantitatively  qualitatively and  the s a m e a s f o r t h o s e y e a r s w h i c h p r o d u c e d the 1966 a n d  1967 a d u l t p o p u l a t i o n s , than a s f i n g e r l i n g s . are approximately  F ^ h y b r i d s do not a p p e a r l e s s a b u n d a n t a s a d u l t s  O n the o t h e r hand, h y b r i d b a c k c r o s s e s  to M y l o c h e i l u s  f i v e t i m e s as abundant a s f i n g e r l i n g s than as adults.  1966  GILL  NET SAMPLES-  ADULTS.  N = . 39  1967  GILL  NET  SAMPLES  - ADULTS  N = 117  1967  DIP  NET  SAMPLES  - FINGERLIN  N = 341  A N A L FIN R A Y S - N U M B E R S g u r e 17.  D i s t r i b u t i o n of a n a l f i n ' r a y s f r o m  ! ; ;  9  ! !  1967 f i n g e r l i n g s a n d f o r .1966 a n d 1967  to "13" f o r adults.  74 DISCUSSION I n t r o g r e s sion, Swamping, and R e p r o d u c t i v e  Isolation  A n d e r s o n a n d H u b r i c h t (1938) d e f i n e d i n t r o g r e s s i o n a s ". . . a n i n f i l t r a t i o n of the g e r m p l a s m of one  s p e c i e s i n t o that of a n o t h e r . "  S t e b b i n s (1959) s t a t e s that t h e r e a r e t h r e e e s s e n t i a l p h a s e s to t h i s p r o c e s s ; (1)  the i n i t i a l f o r m a t i o n of the F ^ h y b r i d s (Z) t h e i r b a c k c r o s s i n g to one  the o t h e r of the p a r e n t a l s p e c i e s , a n d  (3) the n a t u r a l s e l e c t i o n of c e r t a i n  r e c o m b i n a n t types.  The  cases,  o r a b s e n c e of t h e m i s e a s i l y d e m o n s t r a t e d .  the p r e s e n c e  f i r s t two  or  p h a s e s a r e c l e a r cut and,  in m o s t The  t h i r d p h a s e , h o w e v e r , i s not e a s i l y d e m o n s t r a t e d b e c a u s e the p e r m a n e n c e of the r e c o m b i n a n t t y p e s , a n e c e s s a r y f a v o r i n g t h e m , i s d i f f i c u l t to e s t a b l i s h . r e c o m b i n a n t s can p o s s e s s The  one  conclusion ifnatural selection is The  s e l e c t i v e l y advantageous  or innumerable  a l l e l e s of the o t h e r s p e c i e s .  l a t t e r s i t u a t i o n i s k n o w n a s s w a m p i n g a n d t h i s type of i n t r o g r e s s i o n ,  m a s s i v e i n t r o g r e s s i o n , w i l l be d i s c u s s e d f i r s t . mining  The  d i f f i c u l t y of d e t e r -  the p e r m a n e n c e of t h e s e r e c o m b i n a n t s ( h y b r i d  backcrosses)  a r i s e s because phase 1 i s constantly r e c r u i t i n g F j hybrids which i n turn p r o d u c e the 1st g e n e r a t i o n h y b r i d b a c k c r o s s e s a l l " i n t r o g r e s s i n g " genes may  or recombinants.  be e l i m i n a t e d b y the n e x t  Although  generation, it  w i l l a p p e a r that they a r e p e r m a n e n t a d d i t i o n s to the gene p o o l b e c a u s e they a r e c o n t i n u a l l y b e i n g r e p l e n i s h e d . Backcross  They are, however,  transient.  h y b r i d s s h o u l d not be c o n s i d e r e d a s i n t r o g r e s s a n t s u n t i l t h e i r  p e r m a n e n c e has b e e n e s t a b l i s h e d .  E x a m p l e s of h y b r i d  c o n s t a n t l y b e i n g r e c r u i t e d but s u b s e q u e n t l y  backcrosses  e l i m i n a t e d f r o m the p o p u l a t i o n  75 a r e the p e r s i s t e n t h y b r i d z o n e s b e t w e e n the c y p r i n i d s G i l a o r c u t t i i a n d G. m o h a v e n s i s  of southern  C a l i f o r n i a (Hubbs a n d M i l l e r ,  A c r o c h e i l u s aleutaceus and Ptychocheilus oregonense (Stewart,  1943) a n d  in British  Columbia  1966).  U n l e s s the f r e q u e n c y o f v a r i o u s h y b r i d b a c k c r o s s determined,  g e n e r a t i o n s c a n be  i t a p p e a r s d i f f i c u l t to d e t e r m i n e i f s w a m p i n g i s o c c u r r i n g  w h e n i n i t i a l h y b r i d i z a t i o n a n d b a c k c r o s s i n g a r e p r e s e n t u n l e s s ; (1) the s w a m p i n g o f the two g e n e p o o l s i s so n e a r c o m p l e t i o n  that i t i s u n m i s t a k e -  a b l e , o r (2) the d i s t r i b u t i o n s o f the two p a r e n t a l s p e c i e s f o r g i v e n c h a r a c t e r s a r e not s t a t i o n a r y o v e r t i m e o r space. An  a t t e m p t w a s m a d e to d i f f e r e n t i a t e b e t w e e n v a r i o u s  g e n e r a t i o n s to d e t e r m i n e the p e r m a n e n c e o r , i n v e r s e l y ,  backcross  the t r a n s i e n c e  of " i n t r o g r e s s i n g " g e n e s i n M y l o c h e i l u s a n d R i c h a r d s o n i u s  .  Differentiation  b e t w e e n 1st a n d 2nd o r l a t e r g e n e r a t i o n h y b r i d b a c k c r o s s e s  to M y l o c h e i l u s  w a s a c h i e v e d w i t h a f a i r d e g r e e o f p r e c i s i o n b e c a u s e o f the o p p o r t u n i t y to make  such c r o s s e s experimentally.  Second generation h y b r i d  a r e s c a r c e ; o n l y 1 w a s f o u n d i n 249 s u p p o s e d l y For Richardsonius,  pure M y l o c h e i l u s  e v e n 1st g e n e r a t i o n h y b r i d b a c k c r o s s e s  B a s e d o n the f r e q u e n c y  pure  examined.  are scarce  (1. 7 % o f a l l h y b r i d s ) , w h i l e n o 2nd o r l a t e r g e n e r a t i o n h y b r i d w e r e f o u n d i n a s a m p l e o f 178 s u p p o s e d l y  backcrosses  backcrosses  Richardsonius.  of " h y b r i d " p r o t e i n p a t t e r n s a n d the d i s t r i b u t i o n  of a n a l f i n r a y s , the g e n e s of one s p e c i e s w h i c h a r e " i n t r o g r e s s i n g " into the gene p o o l o f the o t h e r , a p p e a r to be e l i m i n a t e d w i t h s u b s e q u e n t b a c k crossing.  If the two p r o t e i n s ( > 4 l o c i ) a r e c o n s i d e r e d r e p r e s e n t a t i v e o f  the two g e n o m e s , i t i s c o n c l u d e d Evidence  76 that s w a m p i n g i s n o t i n p r o g r e s s .  f o r the a b s e n c e o f s w a m p i n g b e t w e e n M y l o c h e i l u s  Richardsonius  was a l s o gathered  f r o m two m o r p h o l o g i c a l  and  characters,  l a t e r a l l i n e s c a l e s a n d the r a t i o o f the p r e d o r s a l to the p r e p e l v i c l e n g t h . I n d i v i d u a l s f r o m S t a v e L a k e j u d g e d to b e p u r e M y l o c h e i l u s a n d R i c h a r d s o n i u s on the b a s i s of a n a l f i n r a y s w e r e c o m p a r e d w i t h p o p u l a t i o n s w h e r e h y b r i d i z a t i o n does not o c c u r ,  o r w h e r e i t c o u l d n o t h a v e o c c u r r e d i n the p a s t  b e c a u s e o f the a b s e n c e o f one of the two s p e c i e s . for  No s i g n i f i c a n t d i f f e r e n c e s  e i t h e r c h a r a c t e r a r e f o u n d b e t w e e n the m e a n s o f the p o p u l a t i o n s  w i t h i n , a n d o u t s i d e , the h y b r i d zone. zone p o s s e s s  In f a c t , M y l o c h e i l u s  from  f r o m the h y b r i d  h i g h e r m e a n l a t e r a l l i n e s c a l e c o u n t s than a l l o p a t r i c p o p u l a t i o n s ,  e x a c t l y o p p o s i t e the e x p e c t e d  result if Richardsonius  genes f o r low scale  n u m b e r s a r e i n t r o g r e s s i n g into M y l o c h e i l u s . Therefore, f o u n d to s u g g e s t  no evidence  either m o r p h o l o g i c a l l y o r b i o c h e m i c a l l y was  swamping between M y l o c h e i l u s and R i c h a r d s o n i u s ,  t h o u g h h y b r i d b a c k c r o s s i n g to M y l o c h e i l u s  i s not uncommon.  even  Reproductive  isolation is maintained. Although  the a b s e n c e o f i n t r o g r e s s i o n o f s e v e r a l l o c i c o n t r o l l i n g b i o -  chemical and morphological  c h a r a c t e r s is used as evidence  against m a s s i v e  i n t r o g r e s s i o n o r s w a m p i n g b e t w e e n the two s p e c i e s , t h e r e i s no  evidence  to r e f u t e the p o s s i b i l i t y that one, o r two, o r m o r e , g e n e s of e a c h s p e c i e s c o u l d n o t h a v e i n t r o g r e s s e d i n t o the o t h e r . entirely plausible, supposedly  This possibility,  though  i s d i f f i c u l t to p r o v e b e c a u s e one d o e s n o t k n o w i f the  i n t r o g r e s s e d c h a r a c t e r w a s a l r e a d y p r e s e n t i n the i n t r o g r e s s e d  species  at a l o w f r e q u e n c y .  D i r e c t i o n a l s e l e c t i o n of p r e e x i s t i n g v a r i a -  b i l i t y c o u l d t h e n p r o d u c e a r e s u l t s i m i l a r to i n t r o g r e s sion. t y p i c m o d i f i c a t i o n of one a s i m i l a r one  Also,  m o r p h o l o g i c a l c h a r a c t e r to i n a d v e r t e n t l y  i n a n o t h e r s p e c i e s c a n n o t be  r u l e d out.  d i r e c t i o n a l s e l e c t i o n of p r e e x i s t i n g v a r i a b i l i t y ,  phenoresembl  Either introgression  or phenotypic modification  c o u l d e x p l a i n the f a c t that the s p a w n i n g t i m e a n d b r e e d i n g s i z e of Mylocheilus  a p p r o x i m a t e s t h o s e of R i c h a r d s o n i u s  o n l y i n the h y b r i d zone.  78 Isolating M e c h a n i s m s  When both h y b r i d i z a t i o n and r e p r o d u c t i v e i s o l a t i o n are present, it i s c l e a r that the h y b r i d s b e t w e e n two disadvantage. and M i l l e r , Nelson,  s p e c i e s m u s t be at a s e l e c t i v e  T h i s i n f e r e n c e has b e e n m a d e b y m a n y w o r k e r s (Hubbs  1943;  1968).  Sibley,  1961;  Mayr,  1963;  Hagen,  1967;  and  O f t e n , the F ^ h y b r i d s a r e v i g o r o u s but s t e r i l e  (Hubbs,  1955) a n d t h i s i s o l a t i n g m e c h a n i s m p r e v e n t s t h o s e g e n e s f r o m p r o g r e s s i n g any f a r t h e r .  H o w e v e r , m a n y e x a m p l e s a r e k n o w n w h e r e the F j  h y b r i d s a p p e a r f u l l y f e r t i l e (Hubbs and Strawn, 1967; a n d N e l s o n ,  1957a;  Hagen,  1968) a n d o t h e r i s o l a t i n g m e c h a n i s m s m u s t c o n t r i b u t e  to the e l i m i n a t i o n of t h e i r g e n e s . o r t h e y do  1956,  E i t h e r the F ^ h y b r i d s f a i l  m a t e but the F £ o r h y b r i d b a c k c r o s s p r o g e n y  to m a t e  are inviable.  E v i d e n c e f o r h y b r i d i n f e r i o r i t y i n f i s h e s o t h e r than h y b r i d s t e r i l i t y i s meager.  M a n y a u t h o r s (Stewart,  h a v e b e e n u n a b l e to d e m o n s t r a t e  1966;  Hagen,  1967;  Nelson,  1968)  h y b r i d i n f e r i o r i t y , an i n f e r i o r i t y which  m u s t e x i s t i f r e p r o d u c t i v e i s o l a t i o n i s to be m a i n t a i n e d b e t w e e n h y b r i d i z i n g species.  H u b b s (1958) h a s p r e s e n t e d s o m e of the b e s t e v i d e n c e f o r h y b r i d  inferiority. lepidum  F ^ h y b r i d b a c k c r o s s e s to E t h e o s t o m a s p e c t a b i l e a n d  possess poorer survival  to h a t c h i n g a n d to the e a r l y  E.  larval  stage t h a n e i t h e r p a r e n t a l s p e c i e s . Isolating m e c h a n i s m s effecting reproductive isolation between M y l o c h e i l u s and R i c h a r d s o n i u s w e r e e x a m i n e d  g e n e r a l l y i n Stave  Lake,  79 B r i t i s h C o l u m b i a , and i n D e v i l s C r e e k i n p a r t i c u l a r . P r e m a t i n g I s o l a t i n g M e c h a n i s m s - B e t w e e n the P a r e n t a l S p e c i e s Although  the p e a k of s p a w n i n g f o r R i c h a r d s o n i u s  l a t e r than f o r M y l o c h e i l u s ,  s e v e r a l days  the o v e r l a p b e t w e e n t h e m i s so g r e a t that  s e a s o n a l i s o l a t i o n c a n n o t be a n e f f e c t i v e b a r r i e r . and  occurs  spatial factors are poorly developed  Likewise,  temporal  isolating m e c h a n i s m s in D e v i l s  C r e e k s i n c e b o t h s p e c i e s u s u a l l y spawn, b e g i n n i n g at dusk, on the r i f f l e a b o v e the l a k e .  A l s o , eggs c o l l e c t e d f r o m an  i n D e v i l s C r e e k y i e l d e d 11. 8 %  first  18" s q u a r e r i f f l e  hybrids, further evidence  area  f o r the i n e f f e c t i v e -  n e s s of s p a t i a l a n d t e m p o r a l m e c h a n i s m s . D a t a on e t h o l o g i c a l i s o l a t i o n b e t w e e n the two n i g h t o b s e r v a t i o n of r i f f l e s  was  near impossible.  a b s e n c e of f i s h c o u l d be d e t e c t e d . whether  It was  species is m e a g e r since Only.the  not d i s c o v e r e d ,  presence  or  therefore,  h y b r i d s w e r e p r o d u c e d by m i x e d s p e c i e s aggregations  r e s u l t of c h a n c e m e e t i n g of g a m e t e s f r o m c o n s p e c i f i c a g g r e g a t i o n s  or as  the  of e a c h  s p e c i e s. F] Hybrid  Viability  D a t a w e r e c o l l e c t e d on the s u r v i v a l of e g g s to h a t c h i n g of v a r i o u s c r o s s e s made under experimental Mylocheilus x Richardsonius  conditions.  R e c i p r o c a l c r o s s e s of  ( F ^ s ) give good egg  s u r v i v a l and a r e  significantly different f r o m parental M y l o c h e i l u s or Richardsonius The  s u r v i v a l of F ^ h y b r i d s was  f i n g e r l i n g stage ( a p p r o x i m a t e l y  not crosses.  a l s o s t u d i e d f r o m the y o l k - s a c f r y to the 60 d a y s ) u n d e r e x p e r i m e n t a l  conditions  a n d the r e s u l t s , a g a i n , do n o t d i f f e r s i g n i f i c a n t l y f r o m p a r e n t a l M y l o c h e i l u s crosses.  80 S o m e d a t a w e r e a l s o g a t h e r e d f r o m the n a t u r a l e n v i r o n m e n t o n the s u c c e s s o f F ^ h y b r i d s r e l a t i v e to that of M y l o c h e i l u s . ill-adapted decrease F^  to the e n v i r o n m e n t ,  then t h e i r r e l a t i v e a b u n d a n c e  b e t w e e n the f i n g e r l i n g a n d a d u l t s t a g e s .  h y b r i d s w e r e included i n this c o m p a r i s o n ,  fin rays were examined.  If F j h y b r i d s a r e  T h e ratio  of a d u l t  should  T o i n s u r e that m a i n l y  o n l y h y b r i d s w i t h 11 a n a l F\  h y b r i d s to a d u l t M y l o c h e i l u s  w a s 0. 0278:1 i n 1966 a n d 0. 041.5:1 i n 1967, w h i l e the r a t i o o f f i n g e r l i n g h y b r i d s to f i n g e r l i n g M y l o c h e i l u s  i n 1967 w a s 0. 0350:1.  A s s u m i n g that the  p r o d u c t i o n o f F ^ h y b r i d s i n 1967 w a s s i m i l a r to those y e a r s w h i c h  resulted  i n the 1966 a n d 1967 a d u l t h y b r i d p o p u l a t i o n s , i t a p p e a r s that, r e l a t i v e to Mylocheilus,  they a r e n o t i n f e r i o r i n s u r v i v a l .  In c o n c l u s i o n , t h e r e f o r e , e g g s u r v i v a l o f Fi h y b r i d s u n d e r  experimental  c o n d i t i o n s i s c o m p a r a b l e to that o f the p a r e n t a l s p e c i e s ; s u r v i v a l of y o l k s a c f r y to the f i n g e r l i n g stage i s e q u a l to M y l o c h e i l u s . ditions,  t h e r e i s no e v i d e n c e  than f o r M y l o c h e i l u s . and  Richardsonius  that the s u r v i v a l o f F ^ h y b r i d s i s any p o o r e r  T h e g o o d v i a b i l i t y o f Fi h y b r i d s b e t w e e n M y l o c h e i l u s  i s i n a g r e e m e n t w i t h the f i n d i n g s o f N e l s o n  Fj h y b r i d s b e t w e e n two s p e c i e s o f c a t o s t o m i d s two  f o r m s ( s p e c i e s ? ) of G a s t e r o s t e u s  there i s no evidence hybrids among fishes  (1968) f o r  and for F ^ hybrids between  r e p o r t e d b y H a g e n (1967).  of h y b r i d v i g o r , a s r e p o r t e d f o r m a n y (i. e. C e n t r a r c h i d a e ,  Hubbs,  Spawning B e h a v i o u r of H y b r i d As  Under natural con-  Also,  interspecific  1955).  Fishes  i n the c a s e o f the p a r e n t a l s p e c i e s the t e c h n i c a l d i f f i c u l t i e s o f  o b s e r v i n g a t n i g h t l i m i t e d o b s e r v a t i o n s of r e p r o d u c t i v e b e h a v i o u r  for  81 hybrid individuals.  However,  s o m e i n f e r e n c e s c a n be m a d e f r o m  and n a t u r a l l y spawned eggs c o l l e c t e d f r o m D e v i l s Creek.  specimens  Hybrids in  s p a w n i n g c o n d i t i o n a r e a b u n d a n t i n g i l l n e t s s e t b e l o w the f i r s t r i f f l e of Devils  C r e e k d u r i n g the s p a w n i n g p e r i o d ; they c o m p o s e d  s p a w n i n g f i s h i n 1966 overestimated  and  11. 5 %  i n 1967.  b e c a u s e the 38 m m  The  of a l l  f o r m e r figure is somewhat  g i l l n e t s u s e d i n 1966  the s m a l l e r s p e c i e s , R i c h a r d s o n i u s .  18. 2 %  discrepancy,  the  p e r c e n t a g e of h y b r i d s i n s p a w n i n g c o l o r a t i o n i n D e v i l s C r e e k i s h i g h ,  and  suggests  the l a c k of t e m p o r a l a n d  the p a r e n t a l s p e c i e s .  Evidence  Regardless  of t h i s  d i s c r i m i n a t e d against  s p a t i a l i s o l a t i o n b e t w e e n the h y b r i d s  c o r r o b o r a t i n g the s u g g e s t i o n that h y b r i d s  a r e not s p a t i a l l y o r t e m p o r a l l y i s o l a t e d f r o m the p a r e n t a l 24. 6 %  and  s p e c i e s i s that  of the f i s h c o l l e c t e d f r o m a s p a w n i n g a g g r e g a t i o n i n a n 18"  square  r i f f l e a r e a iri D e v i l s C r e e k w e r e h y b r i d s . T h e r e i s a l s o a s u g g e s t i o n that at l e a s t s o m e h y b r i d f e m a l e s a r e a b l e to c o m p l e t e s p a w n i n g s u c c e s s f u l l y . (females  The  p e r c e n t a g e of s p e n t  that h a d p r o b a b l y d e p o s i t e d t h e i r e g g s ) i s s i g n i f i c a n t l y h i g h e r  f o r h y b r i d s than f o r M y l o c h e i l u s .  The  p r e s e n c e of n u m e r o u s B C M  f r o m e g g s c o l l e c t e d f r o m the s p a w n i n g r i f f l e s the n u m b e r o u s B C M 1967  a l l i n d i c a t e that s o m e h y b r i d f e m a l e s do These BCM  zone of the l a k e i n  spawn and d e p o s i t t h e i r eggs  m a l e s r a t h e r than  from  females x hybrid males because hybrid males possess  q u a n t i t i e s of a b n o r m a l m i l t and survival. .  and  progeny most likely originate f r o m  c r o s s e s between hybrid f e m a l e s x M y l o c h e i l u s Mylocheilus  progeny  of D e v i l s C r e e k i n 1966  f i n g e r l i n g s c a p t u r e d i n the l i t t o r a l  in an adequate manner.  egg  females  c r o s s e s involving them produce very  small poor  82 F r o m the s c a r c i t y of h y b r i d s w i t h 9 a n a l f i n r a y s (3/56),  a  very  l a r g e p e r c e n t a g e of t h e s e s p a w n i n g h y b r i d s a r e p r e s u m e d to be of 'Fj origin.  Apparently,  Richardsonius  adult h y b r i d b a c k c r o s s e s  to M y l o c h e i l u s  and  a r e a s s c a r c e i n the s p a w n i n g p o p u l a t i o n s a s they a r e i n  s a m p l e s t a k e n f r o m the p e l a g i c a r e a s of the l a k e . In c o n c l u s i o n , t h e r e i s no e v i d e n c e  that the s p a w n i n g b e h a v i o u r  h y b r i d f e m a l e s s e r v e s as an effective i s o l a t i n g m e c h a n i s m . the f a c t that h y b r i d m a l e s a r e not s e a s o n a l l y , t e m p o r a l l y ,  Except for or  spatially  s e g r e g a t e d f r o m the p a r e n t a l s p e c i e s , no i n f o r m a t i o n on t h e i r behaviour  is available.  b e t w e e n the s p e c i e s  A  spawning  possible ethological isolating m e c h a n i s m  w o u l d be the h o m o g a m o u s m a t i n g of h y b r i d s .  s u c h c r o s s e s a r e v e r y u n s u c c e s s f u l , gene f l o w w o u l d c e a s e . spawning behaviour  of  Since  However,  the  of the p a r e n t a l s p e c i e s - - t h e y b o t h s p a w n i n l a r g e ,  t i g h t l y p a c k e d , a g g r e g a t i o n s - - w o u l d p r o b a b l y not a l l o w h y b r i d m a l e s the e x c l u s i v e u s e of a h y b r i d f e m a l e .  F] Hybrid Egg  F e r t i l i t y and H y b r i d B a c k c r o s s  Viability  s u r v i v a l e x p e r i m e n t s i n v o l v i n g h y b r i d m a l e s and f e m a l e s w e r e  p e r f o r m e d to a s c e r t a i n t h e i r f e r t i l i t y r e l a t i v e to the p a r e n t a l f o r m s . Crosses and  involving hybrid females,  Richardsonius  F^s,  and  Mylocheilus  m a l e s a r e not s i g n i f i c a n t l y d i f f e r e n t i n e g g  f r o m p a r e n t a l M y l o c h e i l u s and Apparently,  p r e s u m e d to be  Richardsonius  crosses, respectively.  t h e r e f o r e , h y b r i d f e m a l e s a r e quite f e r t i l e .  hand, c r o s s e s of p r e s u m e d F ^ s p e c i e s y i e l d p o o r egg  survival  On  the o t h e r  h y b r i d m a l e s w i t h f e m a l e s of b o t h p a r e n t a l  survival,  s u g g e s t i n g the p a r t i a l s t e r i l i t y of t h e s e  83 males.  Other evidence  f o r the p a r t i a l s t e r i l i t y  of p r e s u m e d F ^  hybrid  m a l e s i s the a p p e a r a n c e of t h e i r m i l t ; i n c o n t r a s t to m a l e s of the p a r e n t a l s p e c i e s , i t i s t r a n s p a r e n t and v e r y r e d u c e d  i n quantity.  The  sperm,  t h e m s e l v e s , a r e m o r e v a r i a b l e i n s i z e than t h o s e of M y l o c h e i l u s . However,  the b e t t e r egg  s u r v i v a l of h y b r i d b a c k c r o s s e s  with  Richardsonius  than with M y l o c h e i l u s f e m a l e s suggests  that g e n e t i c i n c o m p a t a b i l i t y  may  be a c o n t r i b u t i n g f a c t o r i n the p o o r e g g  s u r v i v a l of c r o s s e s i n v o l v i n g F^  hybrid males. The  m e a n egg  s u r v i v a l of p r e s u m e d F ^ h y b r i d $$ x p r e s u m e d  h y b r i d cfd c r o s s e s i s the p o o r e s t of a l l c r o s s e s a t t e m p t e d . these c r o s s e s c h a r a c t e r i s t i c a l l y p r o d u c e a b n o r m a l fry. only  4 i n d i v i d u a l s w e r e r e a r e d to the f i n g e r l i n g In one  instance, a single M y l o c h e i l u s  Mylocheilus male, Richardsonius  a hybrid backcross  pared  to 81. 4 %  F^ h y b r i d male.  backcrosses  F r o m 632  The  c r o s s e d with a  to M y l o c h e i l u s m a l e ,  to M y l o c h e i l u s  m i l t of the B C M  2  f e r t i l i t y f a c t o r s had o c c u r r e d .  m a l e i s 92. 2 %  and  m a l e was  Obviously,  eggs,  stage.  f e m a l e was  f o r the c o n t r o l ( M y l o c h e i l u s d)  s i m i l a r to m a l e s of b o t h s p e c i e s . Mylocheilus  Additionally,  m a l e s , a n d a h y b r i d m a l e of p r e s u m e d F i o r i g i n .  s u r v i v a l w i t h the h y b r i d b a c k c r o s s  Fj  1. 7 %  Egg com-  f o r the p r e s u m e d  w h i t i s h i n a p p e a r a n c e _and  assortment for parental The  p r o p o r t i o n of h y b r i d  w i t h r e s t o r e d f e r t i l i t y i s p r o b a b l y a f u n c t i o n of the n u m b e r of  genes by w h i c h it i s c o n t r o l l e d . A  s t a t e m e n t s h o u l d be m a d e a b o u t the r e l a t i o n s h i p of e g g  m e a s u r e d e x p e r i m e n t a l l y to that u n d e r n a t u r a l c o n d i t i o n s . E g g  s u r v i v a l as survival  84 u n d e r e x p e r i m e n t a l c o n d i t i o n s i s p r o b a b l y s u b s t a n t i a l l y better than n a t u r a l survival.  T h i s i s b e c a u s e of the way  experimentally.  The  the e g g s a r e f e r t i l i z e d a n d  milt is placed directly  on the e g g s a n d the e g g s  s u b s e q u e n t l y s u b m e r g e d i n a s m a l l q u a n t i t y of w a t e r f o r a b o u t to w a t e r h a r d e n .  15 m i n u t e s  U n d e r the n a t u r a l c o n d i t i o n s of s w i f t r i f f l e s ,  a n d e g g s a r e p r o b a b l y i n c o n t a c t f o r o n l y a v e r y few the p r o b a b i l i t y of f e r t i l i z a t i o n m a y  be r e d u c e d .  the  Dead eggs infected with  r e d u c i n g the l o s s by c o n t a m i n a t i o n o f o t h e r w i s e v i a b l e e g g s .  How  sperm  seconds and therefore  f u n g u s w e r e u s u a l l y r e m o v e d f r o m e x p e r i m e n t a l l o t s e v e r y day,  fungus  treated  thus  In n a t u r e ,  i n f e c t e d e g g s a t t a c h e d to r o c k s i n D e v i l s C r e e k w e r e v e r y abundant.  i m p o r t a n t t h e s e two f a c t o r s a r e i n r e d u c i n g the n u m b e r of o f f s p r i n g  produced  by  h y b r i d m a l e s i s n o t known.  In c o n c l u s i o n , the f e r t i l i t y of F ^ h y b r i d s i s not a n e f f e c t i v e i s o l a t i n g m e c h a n i s m between M y l o c h e i l u s and R i c h a r d s o n i u s  i n Stave  Lake.  H o w e v e r , the p a r t i a l s t e r i l i t y of F ^ h y b r i d m a l e s w o u l d c e r t a i n l y the s p e e d of s w a m p i n g b e t w e e n the two  decrease  species, if other isolating mechan-  i s m s were! n o t e f f e c t i v e . E x p e r i m e n t a l d a t a s h o w that the s u r v i v a l f r o m f i n g e r l i n g stage i s s i g n i f i c a n t l y l e s s (approx. to M y l o c h e i l u s  20%)  the y o l k - s a c f r y to the for hybrid backcrosses  than f o r p a r e n t a l M y l o c h e i l u s i n d i v i d u a l s o r f o r r e c i p r o c a l  F j hybrid individuals. It s h o u l d be u n d e r s t o o d  that t h i s 2 0 %  a p e r i o d of o n l y 48 d a y s - - p r o b a b l y Consequently,  decrease in survival occurs  a s m a l l f r a c t i o n of the n o r m a l l i f e  over span.  s u c h a d i f f e r e n t i a l m o r t a l i t y s u m m e d o v e r the l i f e s p a n of  85 the h y b r i d b a c k c r o s s e s s e e m s a d e q u a t e to e l i m i n a t e v i r t u a l l y a l l o f t h e m , thus i n s u r i n g the m a i n t e n a n c e o f r e p r o d u c t i v e species.  Besides  this p o o r e r s u r v i v a l ,  i s o l a t i o n b e t w e e n the two  h y b r i d b a c k c r o s s e s to M y l o c h e i l u s  c o n t a i n m o r e a b n o r m a l i n d i v i d u a l s (11. 5 % ) than p a r e n t a l M y l o c h e i l u s o r F^  h y b r i d lots.  If the 11. 5 % a b n o r m a l B C M s a r e c o n s i d e r e d  t i e s u n d e r n a t u r a l c o n d i t i o n s , a s they p r o b a b l y  as m o r t a l i -  s h o u l d be, t h i s f u r t h e r  i n c r e a s e s the d i f f e r e n t i a l m o r t a l i t y b e t w e e n t h e m a n d M y l o c h e i l u s Also,  some  s e l e c t i v e f a c t o r s (predation, disease,  p a r a s i t i s m , etc. )  acting against hybrid b a c k c r o s s e s in nature are probably experimental  h o l d i n g tanks.  T o my  to 3 1 % .  a b s e n t f r o m the  k n o w l e d g e , w i t h the e x c e p t i o n o f  H u b b s ' (1958) w o r k o n h y b r i d i z a t i o n i n E t h e o s t o m a ,  t h i s i s the o n l y study  w h e r e the i n f e r r e d i n v i a b i l i t y of h y b r i d b a c k c r o s s p r o g e n y ( e v e n u n d e r l a b o r a t o r y conditions ) has been demonstrated. S o m e c i r c u m s t a n t i a l e v i d e n c e f r o m the h y b r i d z o n e s u g g e s t s that B C M s are  selected against,  r e l a t i v e to M y l o c h e i l u s .  W h e n the n u m b e r of i n d i v i -  d u a l s w i t h 9 a n a l f i n r a y s , w h i c h a r e a s s u m e d to be p r i m a r i l y h y b r i d b a c k c r o s s e s to M y l o c h e i l u s , a r e c o m p a r e d a s f i n g e r l i n g s a n d a s a d u l t s , i t i s a p p a r e n t that f i n g e r l i n g B C M s a r e a p p r o x i m a t e l y f i v e t i m e s a s a b u n d a n t as adults.  Unfortunately,  o n l y one y e a r s  i f t h i s y e a r c a n be c o n s i d e r e d  data a r e a v a i l a b l e .  representative,  However,  then this i s good e v i d e n c e  that B C M s a r e a t a s e l e c t i v e d i s a d v a n t a g e r e l a t i v e to M y l o c h e i l u s .  This  type o f i n f o r m a t i o n n o t o n l y s u g g e s t s that the B C M s a r e s e l e c t e d a g a i n s t , but a l s o i n d i c a t e s what t i m e i n t e r v a l i n t h e i r l i v e s s e l e c t i o n against t h e m is strong.  T h e f i e l d d a t a i n d i c a t e that i n t e n s e s e l e c t i o n o c c u r s  between  86 the f i n g e r l i n g a n d  the a d u l t stage.  In one  way,  this s e e m s  contradictory  b e c a u s e l a b o r a t o r y e x p e r i m e n t s i n d i c a t e that s e l e c t i o n i s s t r o n g f r o m yolk-sac  f r y to the f i n g e r l i n g stage.  a l l s t a g e s of t h e i r l i v e s . identify B C M s before the y o l k - s a c  f r y and  Perhaps,  Because it is impossible  the f i n g e r l i n g stage,  suspect  in nature.  F r o m the f i n g e r l i n g s  i n the l i t t o r a l z o n e of the l a k e i n 1967,  f i n g e r l i n g stage,  (in t h i s s t u d y ) to  f i n g e r l i n g s t a g e s c o u l d be o b t a i n e d  B C R s are numerous.  during  no m e a s u r e of s e l e c t i o n b e t w e e n  D a t a on the v i a b i l i t y of B C R s i s m e a g e r . captured  s e l e c t i o n is intense  the  t h e r e i s no  r e a s o n to  W h e t h e r they a r e e l i m i n a t e d b e f o r e  the  o r a r e n e v e r p r o d u c e d , i s unknown.  W h a t s e l e c t i v e f a c t o r s a r e o p e r a t i n g to m a i n t i a n the i n f e r i o r i t y of h y b r i d b a c k c r o s s e s i s unknown. of the e c o l o g y of the two  As  H a g e n (1967) p o i n t s out,  s p e c i e s i s e s s e n t i a l i f one  s e l e c t i v e f a c t o r s o p e r a t i n g a g a i n s t the h y b r i d s . others,  m e c h a n i s m s which effect reproductive  a knowledge  i s to u n d e r s t a n d the  In t h i s study, a s i n s e v e r a l isolation between  two  s p e c i e s a r e not the o b v i o u s o n e s of h y b r i d s t e r i l i t y o r e t h o l o g i c a l i s o l a t i o n but a p p e a r to be  the c o m p o n e n t o n e s a s s o c i a t e d w i t h the i n f e r i o r i t y of  hybrid backcrosses,  i n part.  C a u s e ( s ) of H y b r i d i z a t i o n a n d The and  L a c k of R e i n f o r c e m e n t  e x c e l l e n t w o r k of C a r l H u b b s (1955  his colleagues  a b o u t the p r o c e s s .  and m a n y e a r l i e r p a p e r s )  on h y b r i d i z a t i o n i n f i s h e s r e v e a l s s e v e r a l g e n e r a l i z a t i o n s One  disrupted environments.  i s the c o r r e l a t i o n b e t w e e n h y b r i d i z a t i o n a n d . The  c a u s e ( s ) of h y b r i d i z a t i o n i n m a n y c a s e s i s  87 apparently  due  to the a l t e r a t i o n of the e n v i r o n m e n t r a t h e r than to a l t e r -  a t i o n s i n the g e n o m e s of the two  h y b r i d i z i n g s p e c i e s . • In Stave L a k e ,  hybridization between M y l o c h e i l u s  and  Richardsonius  is probably related  to the e n l a r g e m e n t of the o r i g i n a l l a k e f o r h y d r o e l e c t r i c p u r p o s e s . original facilities were constructed and  1923.  i n 1911  H y b r i d i z a t i o n b e t w e e n the two  at l e a s t s i n c e 1950,  a p e r i o d of 18  with modifications  in  The 1916  s p e c i e s i n Stave L a k e has  occurred  years.  R o n a l d F i s h e r (1930) i n i t i a l l y p r o p o s e d that n a t u r a l s e l e c t i o n a c t i n g a g a i n s t h y b r i d i n d i v i d u a l s w o u l d i n turn act a g a i n s t those h y b r i d i z i n g i n d i v i d u a l s of e a c h s p e c i e s , "reinforcement  thus f a v o r i n g c o n s p e c i f i c m a t i n g s .  of i s o l a t i n g m e c h a n i s m s " w o u l d , v/ith t i m e ,  e l i m i n a t e h y b r i d i z a t i o n b e t w e e n two is present  species.  i s o l a t i n g m e c h a n i s m s ( S i b l e y , 1961). L i k e w i s e ,  pseudoobscura  D.  also many recorded  persimilis  that h y b r i d s b e t w e e n two  m a c r o c h e i l u s and years.  Various  occurring.  C.  of p r e m a t i n g (1950) has  However, there  are  persisted for many  H u b b s (1961) p r e s e n t s  f o r m s of S i p h a t e l e s o b e s u s  commersonii  or  Drosophila  evidence  have e x i s t e d f o r  S i m i l a r l y , N e l s o n (1968) b e l i e v e s that  In the p r e s e n t  i z e d f o r at l e a s t 18  i n the l a b o r a t o r y .  situations w h e r e h y b r i d i z a t i o n has  y e a r s without r e i n f o r c e m e n t  h u n d r e d s of y e a r s .  Koopman  of i s o l a t i n g m e c h a n i s m s b e t w e e n  and  reduce  M u c h circumstantial evidence  i n the e v o l u t i o n a r y l i t e r a t u r e of r e i n f o r c e m e n t  shown r e i n f o r c e m e n t  This  Catostomus  have h y b r i d i z e d f o r s e v e r a l h u n d r e d  study, M y l o c h e i l u s  and  Richardsonius  have  hybrid-  years.  a u t h o r s s u g g e s t f a c t o r s g o v e r n i n g the s p e e d w i t h w h i c h  88  reinforcement selection  i s completed.  S i b l e y (1961) s t a t e s that " T h e  i n t e n s i t y of  a g a i n s t the h y b r i d s d e t e r m i n e s the s p e e d a n d e x t e n t of r e i n f o r c e -  m e n t of the i s o l a t i n g m e c h a n i s m s . " i n t e n s i t y of s e l e c t i o n ,  (italics mine).  Undoubtedly,  the  " s " ( L i , 1955), i s i m p o r t a n t but f r e q u e n t l y the  i n t e n s i t y of s e l e c t i o n i s 1 ( m a x i m u m i n t e n s i t y of s e l e c t i o n a g a i n s t a g i v e n g e n o t y p e ) b e c a u s e the h y b r i d s a r e s t e r i l e o r i n v i a b l e . persists.  E q u a l l y important,  Yet, h y b r i d i z a t i o n  i t s e e m s , i s the g e n e t i c b a s i s ( n u m b e r of  g e n e s a n d l i n k a g e ) f o r the i s o l a t i n g m e c h a n i s m s t h e m s e l v e s . r e i n f o r c e m e n t w o u l d be c o m p l e t e i n one  For  g e n e r a t i o n i f the h y b r i d s w e r e  s t e r i l e ( s = l ) a n d i f the p a r t i a l l y e f f e c t i v e i s o l a t i n g m e c h a n i s m ( s ) c o n t r o l l e d b y a s i n g l e , d o m i n a n t , gene. O n w o u l d be  required before reinforcement  m e c h a n i s m ( s ) was such cases,  example,  the o t h e r hand, m a n y  was generations  c o u l d be c o m p l e t e d i f the i s o l a t i n g  c o n t r o l l e d m u l t i f a c t o r i a l l y a n d w i t h tight l i n k a g e .  In  t h o s e a l l e l e s w h i c h c o n t r i b u t e to the i n a d e q u a c y of the i s o l a t i n g  m e c h a n i s m ( s ) w o u l d be e x p o s e d to n a t u r a l s e l e c t i o n o n l y i n t e r m i t t e n t l y . C o n s e q u e n t l y , t h e i r e l i m i n a t i o n f r o m the gene p o o l ( s ) w o u l d be may  slow.  be a n e x p l a n a t i o n f o r the l a c k of r e i n f o r c e m e n t b e t w e e n the two  i z i n g s p e c i e s of c a t o s t o m i d s  d e s c r i b e d by N e l s o n  (1968).  He  catostomids et a l . ,  p r o b a b l y do not s p a w n m o r e than two  I966).  However,  the  female  or three times  (Geen  T h u s the i n t e n s i t y of s e l e c t i o n a g a i n s t h y b r i d i z i n g i n d i v i d u a l s  w o u l d be . 50 a n d FjS.  hybridi-  b e c a u s e a m i s m a t e d i n d i v i d u a l has  o p p o r t u n i t y to m a t e c o n s p e c i f i c a l l y i n f u t u r e y e a r s .  hybrid-  believes  that ". . . s e l e c t i o n h a s not b e e n s e v e r e e n o u g h to h a v e p r e v e n t e d z a t i o n i n any k n o w n l o c a l i t y . "  This  . 33,  r e s p e c t i v e l y , s i n c e a l l h y b r i d s a r e e l i m i n a t e d as  T h e s e i n t e n s i t i e s s h o u l d be a d e q u a t e (in the l e n g t h of t i m e that  , h y b r i d i z a t i o n has  8  9  s u p p o s e d l y o c c u r r e d ) to p r o d u c e r e i n f o r c e m e n t ,  i f the  g e n e t i c b a s i s of the i s o l a t i n g m e c h a n i s m s a r e not h i g h l y m u l t i f a c t o r i a l .  It  s e e m s c l e a r that the i n t e n s i t y of s e l e c t i o n i s not the c a u s a t i v e f a c t o r f o r the l a c k of r e i n f o r c e m e n t  i n the c a s e of the h y b r i d i z i n g c a t o s t o m i d s .  M o o r e (1957) s u g g e s t s that l a c k of r e i n f o r c e m e n t two  h y b r i d i z i n g s p e c i e s b e c a u s e r e i n f o r c e d g e n o t y p e s a r e at a s e l e c t i v e  a d v a n t a g e o n l y i n the h y b r i d zone. ing  persists.between  these genotypes,  A l l o p a t r i c populations,  supply n o n - r e i n f o r c e d  m a i n t a i n e d and An  possess-  i n d i v i d u a l s to the h y b r i d  via immigration, which subsequently hybridize. m e c h a n i s m s (postmating) are also present,  not  B e c a u s e other  reproductive  zone  isolating  isolation is  swamping prevented.  a l t e r n a t i v e e x p l a n a t i o n f o r the l a c k of r e i n f o r c e m e n t  z o n e s i s the a b s e n c e of g e n e t i c e a c h s p e c i e s w h i c h do  and  in hybrid  d i f f e r e n c e s b e t w e e n t h o s e i n d i v i d u a l s of  t h o s e w h i c h don't h y b r i d i z e .  In s u c h i n s t a n c e s ,  t h e r e w i l l be no b a s i s on w h i c h r e i n f o r c i n g s e l e c t i o n c a n o p e r a t e . p l a u s i b l e s u c h an explanation  i s , may  How  be k n o w n w h e n the c a u s e s of h y b r i d -  ization are better understood. In s u m m a r y , the p e r s i s t e n c e c a t o s t o m i d s , and  centrarchids  of i n t e r s p e c i f i c h y b r i d s  c a n be i n t e r p r e t e d a s e v i d e n c e f o r e i t h e r :  1) the t i g h t l i n k a g e b e t w e e n , a n d  the l a r g e n u m b e r of, g e n e s c o n t r o l l i n g  p r e m a t i n g isolating m e c h a n i s m s between species, non-reinforced  i n the c y p r i n i d s ,  2) the i m m i g r a t i o n of  g e n o t y p e s i n t o the h y b r i d zone ( M o o r e ' s h y p o t h e s e s ) ,  3) the l a c k of a g e n e t i c b a s i s f o r h y b r i d i z a t i o n .  Since  or  the i n t e n s i t y of.  s e l e c t i o n i s s e v e r e in m a n y c a s e s w h e r e f i s h s p e c i e s have h y b r i d i z e d , i t  90 i s p r o b a b l y o f l i m i t e d i m p o r t a n c e i n the f a i l u r e o f r e i n f o r c e m e n t to o c c u r . F o r M y l o c h e i l u s and R i c h a r d s o n i u s i n Stave L a k e , forcement may  the l a c k of r e i n -  be a t t r i b u t e d to a n y of the a b o v e e x p l a n a t i o n s ,  postulated by M o o r e .  N o n - r e info r e e d genotypes  i n t o the l a k e b e c a u s e of h y d r o e l e c t r i c d a m s . a b s e n c e of r e i n f o r c e m e n t b e t w e e n t h e s e two  s a v e the one  p r o b a b l y cannot  immigrate  A f u r t h e r r e a s o n f o r the s p e c i e s i n Stave L a k e  could  be the s h o r t l e n g t h of t i m e r e i n f o r c i n g s e l e c t i o n h a s b e e n o p e r a t i n g ( 18 years minimum).  W h e t h e r t h i s l e n g t h of t i m e i s a d e q u a t e a g a i n d e p e n d s  o n the g e n e t i c c o n t r o l o f the i s o l a t i n g m e c h a n i s m s t h e m s e l v e s . i n t e n s i t y of s e l e c t i o n i s s u r e l y s t r o n g enough.  i  The  91 SUMMARY The  AND  CONCLUSIONS  p e a m o u t h chub, M y l o c h e i l u s c a u r i a u m  (Richardson)  a n d the  r e d s i d e s h i n e r , R i c h a r d s o n i u s b a l t e a t u s , ( R i c h a r d s o n ) a r e two c l o s e l y r e l a t e d c y p r i n i d f i s h e s i n h a b i t i n g the n o r t h w e s t e r n States and B r i t i s h C o l u m b i a .  United  While m a i n t a i n i n g identities as  s p e c i e s t h r o u g h o u t m o s t of t h e i r s y m p a t r i c  ranges,  they e n g a g e  in extensive h y b r i d i z a t i o n i n Stave L a k e , B r i t i s h  Columbia.  The  if reproductive  purposes  of t h i s study w e r e (1) to d e t e r m i n e  i s o l a t i o n w a s b e i n g m a i n t a i n e d b e t w e e n t h e m a n d (Z) i f i t was,  what  isolating mechanisms were responsible? Two  a p p r o a c h e s w e r e u s e d to a s c e r t a i n the p r e s e n c e  reproductive isolation.  The first,  o r a b s e n c e of  a m o r p h o l o g i c a l one,  attempted  to m e a s u r e a n y s h i f t i n the m e a n s of m o r p h o l o g i c a l c h a r a c t e r s b e t w e e n p o p u l a t i o n s w i t h i n a n d o u t s i d e the h y b r i d zone. s h i f t c o u l d be i n t e r p r e t e d a s r e s u l t i n g f r o m both c h a r a c t e r s used,  Such a  introgression.  For  no s h i f t s i n m e a n s w e r e d e t e c t e d i n e i t h e r  s p e c i e s , i n d i c a t i n g that s w a m p i n g w a s n o t i n p r o g r e s s . The  second approach,  attempted backcross  a c o m b i n e d m o r p h o l o g i c a l - b i o c h e m i c a l one,  to e s t i m a t e the f r e q u e n c y of a d u l t Fx a n d v a r i o u s h y b r i d generations.  If s w a m p i n g w a s i n p r o g r e s s , a d u l t h y b r i d  b a c k c r o s s i n d i v i d u a l s s h o u l d be m o r e n u m e r o u s than a d u l t F j h y b r i e s i n c e t h e r e w o u l d be m a n y m o r e g e n e r a t i o n s of them. backcrosses  to b o t h M y l o c h e i l u s a n d R i c h a r d s o n i u s  n u m e r o u s than a d u l t F ^ h y b r i d s .  Adult hybrid  were much less  A d u l t h y b r i d b a c k c r o s s e s to  92 Richardsonius were very scarce.  Therefore,  s w a m p i n g was  not  s u g g e s t e d by t h i s a p p r o a c h e i t h e r . 5.  In the a b s e n c e of s w a m p i n g , a s e a r c h w a s anism(s)  6.  made for isolating mech-  e f f e c t i n g r e p r o d u c t i v e i s o l a t i o n b e t w e e n the s p e c i e s .  D e v i l s Creek, a s m a l l tributary s t r e a m C o l u m b i a , was  to Stave L a k e ,  u s e d to i n v e s t i g a t e p r e m a t i n g  British  and postmating  isolating  m e c h a n i s m s b e t w e e n the s p e c i e s . 7.  The  s e a s o n a l d i s t r i b u t i o n s of s p a w n i n g M y l o c h e i l u s a n d  a l t h o u g h not s u p e r i m p o s e d extent.  Richardsonius»  u p o n e a c h o t h e r , o v e r l a p to a g r e a t  Spawning h y b r i d s , m a i n l y  F i s , were present  throughout  m o s t of the s p a w n i n g p e r i o d s of b o t h s p e c i e s , a l t h o u g h t h e y  seemed  s o m e w h a t m o r e n u m e r o u s d u r i n g the e a r l y p a r t o f the s p a w n i n g 8.  Not only w e r e both s p e c i e s and  season.  t h e i r h y b r i d s not s e a s o n a l l y i s o l a t e d ,  b u t a l s o they u s u a l l y e n t e r e d D e v i l s C r e e k a t the s a m e t i m e - - a t dusk, o r s h o r t l y t h e r e a f t e r . M o s t o f t e n t h e y a s c e n d e d D e v i l s C r e e k to the f i r s t r i f f l e a b o v e the l e v e l of the l a k e , r e g a r d l e s s of the l a k e l e v e l . Therefore, did 9.  combined temporal  ( s e a s o n a l and diel) and  not a p p e a r to be e f f e c t i v e p r e m a t i n g  Likewise,  i f at a l l .  ( m a i n l y F j s ) w e r e n u m e r o u s i n the s p a w n i n g  comprised  Theoretically, Evidence  isolating mechanisms.  ethological isolation a p p e a r e d p o o r l y developed,  Hybrid females tions and  spatial factors  at l e a s t a n e q u a l p r o p o r t i o n o f a l l spent  populafemales.  they h a d d e p o s i t e d t h e i r e g g s i n a n a d e q u a t e m a n n e r .  to this e f f e c t w a s  the n u m e r o u s h y b r i d b a c k c r o s s  to M y l o c h e i l u s  p r o g e n y (1) i n n a t u r a l l y s p a w n e d e g g s c o l l e c t e d f r o m D e v i l s C r e e k  and  93 ( 2 ) i n n a t u r a l l y p r o d u c e d f i n g e r l i n g s c o l l e c t e d i n the l i t t o r a l z o n e of S t a v e L a k e .  Since F ^ h y b r i d m a l e s have little  p o t e n t i a l (to be  summarized  to M y l o c h e i l u s  p r o g e n y p r o b a b l y a r o s e f r o m h y b r i d r a t h e r than  Mylocheilus 10.  subsequently),  reproductive  these h y b r i d  backcross  females.  E x p e r i m e n t s were, c o n d u c t e d u n d e r l a b o r a t o r y c o n d i t i o n s to t e s t egg  survival.  In g e n e r a l , e g g  c o m b i n a t i o n s of M y l o c h e i l u s ,  s u r v i v a l was  good f o r a l l p o s s i b l e  Richardsonius,  and p r e s u m e d  F^  h y b r i d s e x c e p t f o r c r o s s e s i n v o l v i n g the p r e s u m e d F ^ h y b r i d m a l e . .'Crosses i n v o l v i n g t h i s m a l e y i e l d e d p o o r s u r v i v a l , p a r t l y b e c a u s e of h y b r i d s t e r i l i t y ( s m a l l q u a n t i t i e s of a b n o r m a l m i l t , v a r i a b l y s i z e d s p e r m ) but p a r t l y b e c a u s e of i n v i a b i l i t y f a c t o r s . i n v o l v i n g h y b r i d f e m a l e s and  Crosses  the p a r e n t a l s p e c i e s ' m a l e s gave g o o d  s u r v i v a L .Although p r e s u m e d F ^ h y b r i d m a l e s a r e at l e a s t p a r t i a l l y Sterile,  s w a m p i n g of the s p e c i e s  w o u l d mot be preven-ted, o n l y  slowed  down, b e c a u s e the h y b r i d f e m a l e s w o u l d p r o v i d e an a l t e r n a t e r o u t e . 11.  The  v i a b i l i t y of f r y to the f i n g e r l i n g s t a g e was  experimental swamping.  c o n d i t i o n s and The  Mylocheilus.  fry.  g a v e the o n l y c l u e f o r the a b s e n c e of  s u r v i v a l of r e c i p r o c a l F i  f r y was  H o w e v e r , the h y b r i d b a c k c r o s s  gave a p p r o x i m a t e l y  also tested under  2 0 % poorer  a s g o o d as f o r  to M y l o c h e i l u s f r y  s u r v i v a l than p a r e n t a l M y l o c h e i l u s  A d d i t i o n a l l y , the r e m a i n i n g  a t i o n of the e x p e r i m e n t p o s s e s s e d  fingerlings  at the  termin-  11. 5 % abnormalities.  l e s s s u r v i v a l i n i t s e l f w o u l d not p r e v e n t  While 2 0 %  swamping, it should  be  94 considered days.  that s u c h a d i f f e r e n t i a l o c c u r r e d o v e r a p e r i o d of o n l y  T h i s study i s one  of n a t u r a l h y b r i d s ,  of the few  inferiority  o t h e r than s t e r i l i t y , has b e e n d e m o n s t r a t e d ,  e v e n i f done so u n d e r e x p e r i m e n t a l 12.  i n w h i c h the i n f e r r e d  48  conditions.  Introgre ssion, swamping, and isolating m e c h a n i s m s are  discussed.  95 LITERATURE  A n d e r s o n , E. III.  and  L.  The  Hubricht.  evidence  CITED  1938.  Hybridization in Tradescantia.  for introgre ssive hybridization.  Amer.  J. Bot. , 25_: 396-402. B i gelow, R.  1965.  Hybrid  Evolution, Carl,  z o n e s and  reproductive isolation.  19_(4);449-45 8.  G. C. , W. A.  C l e m e n s , and  C. C.  Lindsey.  w a t e r f i s h e s of B r i t i s h C o l u m b i a .  1959-  The  H a n d b o o k No.  fresh-  5.  British  Columbia Provincial Museum. C o l o w i c k , S. P.  a n d N. O.  Kaplan.  Academic Press, D o b z h a n s k y , T,  1951.  R. A.  1930.  Press,  1963.  Methods in enzymology.  The  VI.  York.  G e n e t i c s a n d the o r i g i n of s p e c i e s .  University Press, Fisher,  New  New  Columbia  York.  g e n e t i c a l t h e o r y of n a t u r a l s e l e c t i o n .  Clarendon  Oxford.  G e e n , G. H. , T. G.  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