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A study of hybridization between two species of cyprinid fishes, Acrocheilus alutaceus and Ptychocheilus… Stewart, Kenneth 1966

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A STUDY OF HYBRIDIZATION BETWEEN TWO SPECIES OF CYPRINID FISHES, ACROCHEILUS ALUTACEUS AND PTYCHOCHEILUS OREGONENSIS  by  KENNETH WILLIAM STEWART B.Sc,  Colorado S t a t e U n i v e r s i t y ,  1958  M . S c , U n i v e r s i t y o f Miami, 1960  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Zoology  We a c c e p t t h i s t h e s i s as conforming t o the r e q u i r e d standard  The U n i v e r s i t y o f B r i t i s h June, 1966  Columbia  In requirements Columbia, for  presenting  this  thesis  in p a r t i a l  f o r an a d v a n c e d  degree  at  I agree  r e f e r e n c e and  tensive  copying  that  the L i b r a r y  study.  I  this  thesis  of  by t h e Head o f my D e p a r t m e n t understood cial  gain  Department  that copying shall  of  not  or  it  1966  freely  purposes  this  thesis  the  British available for  may be  representatives.  be a l l o w e d w i t h o u t my w r i t t e n  Columbia  of  that permission  scholarly  by h i s  Zoology  30 J u n e  s h a l l make  or publication of  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, C a n a d a Date  the U n i v e r s i t y  f u r t h e r agree for  f u l f i l m e n t of  for  It  exgranted  is  finan-  permission.  The U n i v e r s i t y of B r i t i s h  Columbia  FACULTY OF GRADUATE STUDIES  PROGRAMME OF THE . FINAL ORAL EXAMINATION FOR THE DEGREE  OF  DOCTOR OF PHILOSOPHY  of  KENNETH WILL JAM STEWART B.Sc,  Colorado. S t a t e U n i v e r s i t y , 1958  M.Sc,  U n i v e r s i t y o f Miami, 1960  WEDNESDAY., JUNE 29, 1966, AT 3:30 P.M.. IN ROOM 3332, BIOLOGICAL SCIENCES BUILDING  COMMITTEE IN CHARGE Chairman: I . McT. Cowan I. E. E f f o r d W. S. Hoar C. C. L i n d s e y  H. Norden T. G. N o r t h c o t e C. P. S. T a y l o r  E x t e r n a l Examiner: - R. R. M i l l e r Museum of V e r t e b r a t e University  Zoology  of M i c h i g a n  Research S u p e r v i s o r : C. C. L i n d s e y  A STUDY OF HYBRIDIZATION BETWEEN TWO SPECIES OF GYPRINID FISHES, AGROGHEILUS ALUTAGEUS AND PTYCHOCHEILUS OREGONENSIS ABSTRACT Fish morphologically  i n t e r m e d i a t e between Acro-  c h e i l u s a l u t a c e u s and P t y c h o c h e i l u s been c o l l e c t e d system.  i n f r e q u e n t l y from the Columbia  Morphological  P t y c h o c h e i l u s , and and  intra-  oregonensis  have  River  comparisons of w i l d A c r o c h e i l u s ,  intermediates, with a r t i f i c i a l  species crosses i n d i c a t e d that w i l d  inter-  inter-  mediates were of h y b r i d o r i g i n . Observations  &  of spawning habitat; and b e h a v i o r  of  the parent  s p e c i e s suggest: that: h y b r i d i z a t i o n , i n most  localities  is accidental.  In M i s s e z u l a Lake an u n u s u a l l j  h i g h i n c i d e n c e of h y b r i d s , pronounced s p a t i a l of spawning groups of the p a r e n t a l s p e c i e s and of s t r a y P t y c h o c h e i l u s suggest  duce f e r t i l e  are l a r g e l y  sterile,  sperm i n f r e q u e n t l y .  show no evidence  and by  tility  the r a r i t y and  selection against  The  occurs. but. h y b r i d males proThe  of h y b r i d s suggest  partial  sterility  of h y b r i d s  backcrosses. and  partial  fer-  a l a r g e amount of g e n e t i c  s i m i l a r i t y between A c r o c h e i l u s and c o n t e n t i o n i s strengthened  intermediates  Gene flow i s p r o b a b l y  presence of w i l d b a c k c r o s s e s  morphology and  parent, s p e c i e s  of gene flow, but a few  are a p p a r e n t l y b a c k c r o s s e s . b l o c k e d by  presence  i n the s c h o o l of A c r o c h e i l u s a l l  that: i n t e r s p e c i f i c mating  Hybrids  separation  by  Ptychocheilus.  similarity  This  i n chromosome  the p r e s e n c e of dominance e f f e c t s  i n the  i n h e r i t a n c e of some p a r e n t a l c h a r a c t e r s i n h y b r i d s .  GRADUATE STUDIES Field /  of Study:  Ichthyology  Ichthyology Advanced  J, N.  Ichthyology  Problems i n I c h t h y o l o g y  C.  Briggs  J . Wilimovsky C. C,  Lindsey  W.  S. Hoar  Comparative P h y s i o l o g y Other S t u d i e s : H i s t o r y of Fisheries  Philosophy Law  G.  B.  Savery  F.  Curtis  PUBLICATIONS  Stewart,  K.W.,  1962.  Observations  on the morphology  o p t i c a l p r o p e r t i e s of the adipose J. Fish Peden, A.E.,  Res.  Bd. Canada 19;  and K.W.  Stewart.,  Copeia  1964;  e y e l i d of f i s h e s .  1161-1162.  1964.  known range of the a t h e r i n i d affinis.  and  E x t e n s i o n of the  Fish  239-240  Atherinops  KENNETH W I L L I A M STEWART. SPECIES  OF C Y P R I N I D  A STUDY  FISHES,  OF H Y B R I D I Z A T I O N  B E T W E E N TWO  A C R O C H E I L U S A L U T A C E U S AND  j_  PTYCHOCHEILUS  OREGONENSIS  ABSTRACT Supervisor:  C.  C.  Lindsey.  F i s h m o r p h o l o g i c a l l y i n t e r m e d i a t e between A c r o c h e i l u s a l u t a c e u s and P t y c h o c h e i l u s oregonensis  have  been c o l l e c t e d i n f r e q u e n t l y from the Columbia R i v e r system. Morphological and  comparisons of w i l d A c r o c h e i l u s , P t y c h o c h e i l u s ,  intermediates, with a r t i f i c i a l  i n t e r - and  intra-  species  c r o s s e s i n d i c a t e d t h a t w i l d i n t e r m e d i a t e s were o f h y b r i d origin. Observations  o f spawning h a b i t a t and  o f the p a r e n t s p e c i e s suggest localities  is accidental.  behavior  t h a t h y b r i d i z a t i o n i n most  In M i s s e z u l a Lake an  unusually  h i g h i n c i d e n c e o f h y b r i d s , pronounced s p a t i a l s e p a r a t i o n o f spawning groups of the p a r e n t a l s p e c i e s and presence o f s t r a y P t y c h o c h e i l u s i n the s c h o o l of A c r o c h e i l u s a l l suggest that  i n t e r s p e c i f i c mating o c c u r s . Hybrids  are l a r g e l y s t e r i l e , b u t h y b r i d males  produce f e r t i l e sperm i n f r e q u e n t l y . show no evidence  The p a r e n t  of gene flow, but a few  apparently backcrosses.  species  intermediates  Gene flow i s p r o b a b l y b l o c k e d  the r a r i t y and p a r t i a l s t e r i l i t y o f h y b r i d s and by against  are  selection  backcrosses. The presence o f w i l d backcrosses  f e r t i l i t y o f h y b r i d s suggest  and  partial  a l a r g e amount o f g e n e t i c  s i m i l a r i t y between A c r o c h e i l u s and P t y c h o c h e i l u s . c o n t e n t i o n i s strengthened  by  This  by s i m i l a r i t y i n chromosome  morphology and the presence o f dominance e f f e c t s i n the  11  i n h e r i t a n c e of some p a r e n t a l c h a r a c t e r s  in hybrids.  iii ACKNOWLEDGEMENTS I wish t o thank Dr. C.C. L i n d s e y f o r h i s h e l p and encouragement w i t h a l l aspects o f the problem, and f o r h i s h e l p i n r e a d i n g and c o r r e c t i n g o f the manuscript.  Dr. N.J.  Wilimovsky and Dr. W.S. Hoar gave v a l u a b l e a d v i c e on the problem and read and c o r r e c t e d the manuscript.  Thanks a r e  a l s o due t o Dr. P.M. Townsley and Dr. C.V. Finnegan f o r t h e i r h e l p w i t h the c y t o l o g i c a l s t u d i e s which were p a r t o f t h i s work. of  Dr. J.D. McPhail was h e l p f u l w i t h a l l aspects  the c y t o l o g i c a l s t u d i e s , and e s p e c i a l l y f o r p r o v i d i n g  i n s t r u c t i o n i n a technique he developed  for preparing  b r a n c h i a l e p i t h e l i a l c e l l s f o r chromosome; s t u d i e s .  I am  e s p e c i a l l y indebted t o Dr. H. Tsuyuki, who r a n the myogen electropherograms  f o r me.  Many people  a s s i s t e d i n f i e l d o b s e r v a t i o n s and  c o l l e c t i o n s , b u t I e s p e c i a l l y wish t o thank the f o l l o w i n g people. work.  Mr. E.E. Moodie a s s i s t e d i n almost a l l the f i e l d Mr. A l l a n G i l l a l s o p r o v i d e d h e l p and encouragement  i n the f i e l d .  Mr. and Mrs.  G.S. Moss p r o v i d e d u n r e s t r i c t e d  use o f t h e i r f a c i l i t i e s a t M i s s e z u l a Lake. F i n a l l y , my w i f e a s s i s t e d me i n much o f t h e f i e l d work, i n t h e l a b o r a t o r y work, and i n p r e p a r a t i o n o f the manuscript.  iv TABLE OF CONTENTS Abstract  i  Acknowledgements  i i i  T a b l e o f Contents  iv  L i s t o f Tables  v  L i s t o f Figures  vi  Introduction  1  Methods  7  The Study Areas  7  F i e l d C o l l e c t i o n s and Observations  ...  12  Rearing Experiments  14  M o r p h o l o g i c a l S t u d i e s ..  17  E l e c t r o p h o r e t i c Studies  19  C y t o l o g i c a l Studies  20  Results  ..  23  Morphology and E l e c t r o p h o r e s i s  23  R e l a t i v e Abundance o f Hybrids  33  F e r t i l i t y and V i a b i l i t y  40  Cytology  43  Spawning H a b i t a t and Behavior  50  Discussion  55  O r i g i n o f the W i l d Intermediates  55  Causes o f H y b r i d i z a t i o n  58  Consequences o f H y b r i d i z a t i o n  63  R e l a t i o n s h i p o f P t y c h o c h e i l u s and A c r o c h e i l u s ...  67  Summary  70  Literature cited  72  V  LIST OF TABLES Table I II  III IV V  Page Frequency o f p h a r y n g e a l t o o t h counts i n w i l d and r e a r e d p a r e n t a l s p e c i e s and h y b r i d s .  28  Frequency o f o c c u r r e n c e o f bands i n myogen electropherograms o f w i l d p a r e n t a l species and h y b r i d s . .  37  S p e c i e s c o m p o s i t i o n o f p o o l e d c a t c h from M i s s e z u l a Lake and Wolfe Lake  39  Frequency o f d i p l o i d chromosome numbers f o r w i l d p a r e n t a l s p e c i e s and r e a r e d Fj_ h y b r i d s . .  46  Chromosome arm numbers o b s e r v e d f o r w i l d p a r e n t a l s p e c i e s from Wolfe Lake and the Chehalis River  49  vi LIST OF FIGURES Figure 1.  2.  Page D i s t r i b u t i o n o f A c r o c h e i l u s a l u t a c e u s and P t y c h o c h e i l u s o r e g o n e n s i s showing known hybrid l o c a l i t i e s  4  M i s s e z u l a L a k e , showing spawning l o c a l i t i e s f o r A c r o c h e i l u s and P t y c h o c h e i l u s  8  3.  Wolfe Lake and Wolfe Creek  11  4.  L a t e r a l and v e n t r a l p r o f i l e s o f A c r o c h e i l u s , P t y c h o c h e i l u s and w i l d i n t e r m e d i a t e s  24  5.  Numbers o f d i a g o n a l s c a l e rows i n w i l d and r e a r e d A c r o c h e i l u s , P t y c h o c h e i l u s and intermediates  25  Diagrams o f gut c o i l i n g p a t t e r n s f o r Acrocheilus, Ptychocheilus, wild interm e d i a t e s , and j u v e n i l e A c r o c h e i l u s  26  R e l a t i o n s h i p s o f v a r i o u s body p r o p o r t i o n s to standard length i n w i l d A c r o c h e i l u s , P t y c h o c h e i l u s and i n t e r m e d i a t e s  27  8.  Myogen e l e c t r o p h e r o g r a m s o f A c r o c h e i l u s alutaceus . .  34  9.  Myogen e l e c t r o p h e r o g r a m s o f oregonensis  35  6.  7.  Ptychocheilus .  10.  Myogen e l e c t r o p h e r o g r a m s o f w i l d mediates  inter-  11.  C o l c h i c i n e t r e a t e d m i t o t i c metaphase from cultured t e s t i s of Acrocheilus alutaceus . . . .  44  12.  C o l c h i c i n e t r e a t e d m i t o t i c metaphase from Ptychocheilus oregonensis cultured t e s t i s . . .  45  13.  C o l c h i c i n e t r e a t e d m i t o t i c metaphases from b r a n c h i a l e p i t h e l i u m o f r e a r e d F-^ h y b r i d s . . .  48  36  1 INTRODUCTION Mayr d e f i n e s a s p e c i e s as a p o p u l a t i o n o r group o f p o p u l a t i o n s which are r e p r o d u c t i v e l y i s o l a t e d from a l l other populations.  T h i s i s an o b j e c t i v e b u t n o t e n t i r e l y  unambiguous d e f i n i t i o n .  In nature,  t h e r e i s a continuum  between f r e e l y i n t e r b r e e d i n g p o p u l a t i o n s and t h o s e w h i c h occur together without h y b r i d i z a t i o n . Mechanisms a f f e c t i n g the o c c u r r e n c e and r a t e o f gene f l o w between p o p u l a t i o n s have r e c e i v e d a t t e n t i o n many i n v e s t i g a t o r s , Epling  (1947)  from  e s p e c i a l l y i n the f i e l d o f b o t a n y .  i n v e s t i g a t e d the e v i d e n c e f o r gene f l o w  between p l a n t s p e c i e s  in California.  Anderson (1949 and  1953) e x t e n s i v e l y r e v i e w e d i n t r o g r e s s i v e h y b r i d i z a t i o n i n plants. Mayr (1963) summarized knowledge r e g a r d i n g gene flow i n animal p o p u l a t i o n s ,  and many subsequent a u t h o r s have  m o d i f i e d o r e l a b o r a t e d upon h i s v i e w s .  Bigelow  (1965)  re-examines the concepts o f r e p r o d u c t i v e i s o l a t i o n and gene flow i n animal s p e c i e s . G e n e r a l l y these authors conclude that i f popu l a t i o n s are c l o s e l y r e l a t e d g e n e t i c a l l y and t h e r e i s no s e l e c t i o n a g a i n s t h y b r i d s swamping w i l l r e s u l t , r i s e t o a p o p u l a t i o n which combines the c h a r a c t e r i s t i c s of both parental types.  giving  adaptive Introgression  o c c u r s when one o r a few genes o r gene complexes i n t r o duced i n t o the p a r e n t a l p o p u l a t i o n b y h y b r i d i z a t i o n a r e  2  a d a p t i v e l y s u p e r i o r t o the o r i g i n a l genes or complexes. A t the o p p o s i t e extreme, o n l y r a r e , s t e r i l e h y b r i d s may produced, or t h e r e may  be  be no h y b r i d i z a t i o n a t a l l between  r e l a t e d p o p u l a t i o n s i n zones o f c o n t a c t . P r i o r to mating, b e h a v i o r a l , temporal and i s o l a t i o n of b r e e d i n g p o p u l a t i o n s may hybridization. zygote  l i m i t or  spatial  prevent  A f t e r mating, g e n e t i c f a c t o r s c a u s i n g  inviability  or h y b r i d s t e r i l i t y can prevent  from s u r v i v i n g o r mating.  hybrids  I f h y b r i d to h y b r i d matings or  b a c k c r o s s i n g do occur, many o r a l l o f the progeny w i l l s e l e c t e d a g a i n s t because recombination  be  of the p a r e n t a l  genotypes w i l l l e a v e them w i t h unbalanced and p o o r l y adapted genotypes. Compared with other animals, frequently.  f i s h hybridize rather  Hubbs (1955) p o i n t s out t h a t h y b r i d s are more  common i n freshwater  than i n marine f i s h e s ,  and b o r e a l areas than  i n the t r o p i c s , and  than i n s p e c i o s e f i s h  faunas.  i n temperate  i n depauperate  The North American c y p r i n i d s are among the more thoroughly  s t u d i e d groups of f i s h e s w i t h regard to n a t u r a l  hybridization.  Hubbs, alone and w i t h s e v e r a l co-workers  has done much o f t h i s work.  Gilbert  the r e l a t i o n s h i p o f N o t r o p i s cornutus chrysocephalus of  the two  chrysocephalus  (1961) i n v e s t i g a t e d and  Notropis  i n s e v e r a l zones o f c o n t a c t  s p e c i e s of Michigan.  In l a r g e p a r t ,  the  e x i s t i n g s t u d i e s of c y p r i n i d h y b r i d i z a t i o n have been c o n f i n e d  3 to  a n a l y s i s of f i e l d c o l l e c t i o n s of the p a r e n t s  A r t i f i c i a l l y produced F i h y b r i d s i n the  intermediates. etheostomatine  f i s h e s have been r e a r e d i n the l a b o r a t o r y by  Hubbs (1957, 1958 and  1933)  and  and  1959), Hubbs and Hubbs (1932a, 1932b  and have reared Fj_ a r t i f i c i a l c r o s s e s o f Lepomis  s p e c i e s and M o l l i e n e s i a l a t i p i n n a x M.  mexicana.  H y b r i d i z a t i o n among the P a c i f i c Northwestern c y p r i n i d genera was  f i r s t r e p o r t e d by Hubbs and  Schultz  (1931) , f o r A c r o c h e i l u s alutaceus. x P t y c h o c h e i l u s Weisel  (1954, 1955a and 1955b) r e p o r t e d  i n v o l v i n g Richardsonius, Rhinichthys.  oregonensis.  combinations  Ptychocheilus, Mylocheilus  and  A l l of these s t u d i e s were based s o l e l y upon  morphological! a n a l y s i s o f w i l d specimens. The|present study d e a l s with h y b r i d i z a t i o n between s p e c i e s of two nominal genera o f c y p r i n i d squawfish,  P t y c h o c h e i l u s oregonensis  chiselmouth,  Acrocheilus alutaceus  P t y c h o c h e i l u s oregonensis  f i s h e s , the  (Richardson)  northern  and  the  (Agassiz and P i c k e r i n g ) .  and A c r o c h e i l u s a l u t a c e u s both  abundant i n the Columbia R i v e r system.  Ptychocheilus also  occurs throughout the F r a s e r and Skeena R i v e r systems, i n a l l b u t a few Columbia. occurrence  are  c o a s t a l streams o f Washington and  and  British  A c r o c h e i l u s , however, i s s c a t t e r e d i n i n B r i t i s h Columbia, with the e x c e p t i o n o f  Okanagan R i v e r  ( F i g . 1) .  s p e c i e s have been taken  Intermediates  the  between these  i n f r e q u e n t l y s i n c e 1905  Columbia R i v e r system i n the U n i t e d S t a t e s .  two  i n the  Snyder  (1905  4  Fig„  1,  D i s t r i b u t i o n o f A c r o c h e i l u s a l u t a c e u s and P t y c h o c h e i l u s o r e g o n e n s i s showing known h y b r i d localities.  M  100  Km.  H 100 m i l e s A Hybrid L o c a l i t i e s •  Discontinuous  Acrocheilus Localities  —  Boundary  of C o n t i n u o u s A c r o c h e i l u s D i s t r i b u t i o n  *-*  Boundary  of P t y c h o c h e i l u s o r e g o n e n s i s  Distribution  5 and  1908)  recorded an i n t e r m e d i a t e as b e i n g e q u i v a l e n t t o  the L e u c i s c u s c a u r i n u s o f Richardson Schultz  (1836).  Hubbs and  (1931) on the b a s i s o f Snyder's specimen and some  new  m a t e r i a l a t t r i b u t e d t h i s form t o h y b r i d i z a t i o n between A c r o c h e i l u s and P t y c h o c h e i l u s .  S c h u l t z and S c h a e f f e r  and S c h u l t z and De Lacey (1936) r e f e r r e d to a  combination  between A c r o c h e i l u s and M y l o c h e i l u s , c i t i n g Hubbs and (1931).  This, i n fact,  combination. u n t i l Patten  No  (1936)  Schultz  i s the A c r o c h e i l u s x P t y c h o c h e i l u s  subsequent r e f e r e n c e s to t h i s form appeared  (1960) r e p o r t e d i t s occurrence  i n the Yakima  R i v e r , Washington. The p r e s e n t study has  attempted f i r s t t o v e r i f y  t h a t the w i l d i n t e r m e d i a t e s are the r e s u l t o f h y b r i d i z a t i o n . To t h i s end,  morphological  comparisons were made f o r w i l d and  r e a r e d f i s h o f both p a r e n t a l s p e c i e s and probable  the h y b r i d s .  The  causes of h y b r i d i z a t i o n were s t u d i e d by means o f  f i e l d o b s e r v a t i o n s o f the spawning h a b i t a t and b e h a v i o r the p a r e n t a l s p e c i e s .  A l s o , gamete l i f e was  studied i n  order to determine the p o s s i b i l i t y o f h y b r i d i z a t i o n due chance meeting o f d r i f t i n g gametes.  Finally,  of  to  the p o s s i b i l -  i t y o f gene flow between the p a r e n t a l s p e c i e s was  investigated.  The p a r e n t a l s p e c i e s and h y b r i d s were compared f o r a number of  morphological  pherograms.  c h a r a c t e r s , and  f o r muscle e l e c t r o -  Chromosome number and morphology i n the  p a r e n t s and h y b r i d s were s t u d i e d , and h y b r i d f e r t i l i t y the e x i s t e n c e of n a t u r a l l y o c c u r r i n g b a c k c r o s s e s  were  and  6  investigated.  Comparisons o f the r e l a t i v e abundances o f  h y b r i d s t o p a r e n t a l s p e c i e s were made i n o r d e r t o determine the p o s s i b i l i t y o f a s i g n i f i c a n t amount o f gamete wastage due  to h y b r i d i z a t i o n .  7  METHODS A. The Study Areas Missezula and Wolfe Lakes and t h e i r i n l e t and o u t l e t streams were the primary study areas.  Both of these  lakes drain into the Columbia River system v i a the Similkameen and Okanogan Rivers.  The remaining l o c a l i t i e s ,  from which samples of juvenile and adult f i s h were collected, are the following. 1.  Vidette Lake, which drains into the Fraser River system v i a the Thompson River.  2.  Lome and I z z i t s Lakes, which are r e s p e c t i v e l y the uppermost and middle lakes i n the Wolfe Creek drainage which also includes Wolfe Lake.  3.  The Okanogan River, between the towns of Riverside and Brewster, Washington.  1  Missezula Lake i s located 25 miles by road north of Princeton, B r i t i s h Columbia.  I t drains into the Similkameen  River v i a Summers Creek and A l l i s o n Creek.  Missezula i s  the larger of the two i n t e n s i v e l y studied lakes, being 7.3 Km. long, a maximum of 600 M. wide and a maximum depth of 65.3 M. (Fig. 2).  There are several streams draining  into Missezula Lake, only one of which, Leonard Creek, flows constantly.  Leonard Creek i s p a r t l y obstructed by beaver  8  F i g . 2.  M i s s e z u l a Lake, showing spawning l o c a l i t i e s f o r A c r o c h e i l u s and P t y c h o c h e i l u s .  9  dams near i t s mouth and i s q u i t e c o l d . was recorded i n l a t e June a t 9 . 4 ° C .  I t s temperature No c y p r i n i d s were ever  seen spawning i n the f l o w i n g p a r t o f Leonard Creek,  although  R i c h a r d s o n i u s b a l t e a t u s was observed i n spawning c o n d i t i o n in  the shallow, weedy a r e a o f the l a k e around  i t s mouth.  S i n c e none o f the o t h e r creeks were f l o w i n g d u r i n g the time i n which  c y p r i n i d s were i n spawning c o n d i t i o n  ( l a t e May t o  e a r l y J u l y ) Leonard Creek o f f e r s t h e o n l y o p p o r t u n i t y f o r inlet  spawning. The o u t l e t o f M i s s e z u l a Lake i s b l o c k e d by a low  e a r t h dam which r a i s e s the l e v e l o f t h e l a k e b y a minimum o f 1.2 M.  The dam has a l s o had t h e e f f e c t o f a l t e r i n g the  narrow area immediately above i t from a shallow g r a d i e n t stream bed t o an a r e a o f s t a n d i n g water up t o 1.8 M. i n depth.  Rounded stream worn stones c o u l d be found i n the  c e n t e r o f t h i s a r e a beneath lated s i l t . of  There i s a d i r e c t i o n a l c u r r e n t a l o n g the shores  the o u t l e t a r e a which a t t a i n s up t o 15 cm. p e r second  velocity. g r a v e l which It  a p p r o x i m a t e l y 60 cm. o f accumu-  The bottom near the shore c o n s i s t s o f angular i s kept f r e e o f s i l t ,  p r o b a b l y b y the c u r r e n t .  i s i n t h i s a r e a t h a t A c r o c h e i l u s and i n t e r m e d i a t e s i n  spawning c o n d i t i o n were taken.  The dam does not con-  s t i t u t e a b a r r i e r t o f i s h movement, e i t h e r i n t o o r o u t o f the l a k e .  E a s t e r n brook t r o u t were observed p a s s i n g i n  both d i r e c t i o n s i n o v e r f l o w channels around t h e s p i l l w a y . No c y p r i n i d s were e i t h e r c o l l e c t e d o r observed i n the stream  10 below the dam  d u r i n g the study however, so i t may be assumed  t h a t a l l spawning  f i s h are c o n f i n e d t o the l a k e p r o p e r or the  r e g i o n immediately above the dam  ( F i g . 2).  Wolfe Lake i s 10 m i l e s by road e a s t o f P r i n c e t o n . I t l i e s near the mouth o f a v a l l e y opening i n t o the Similkameen canyon, and d r a i n s i n t o the Similkimeen R i v e r v i a a segment o f Wolfe Creek about 1.6 Km. roughly c i r c u l a r , : about 600 M. p r o b a b l y does n o t exceed 6.1 M.  long.  Wolfe Lake i s  i n diameter, and the depth ( F i g . 3).  Wolfe Lake has  a s i n g l e i n l e t stream, Wolfe Creek, which c o n t i n u e s below the l a k e as the o u t l e t .  No o t h e r s u r f a c e d r a i n a g e e i t h e r e n t e r s  or  The i n l e t reached a temperature o f  l e a v e s the l a k e .  19.4°C.  i n l a t e June, o n l y 1 degree c o o l e r than the l a k e  s u r f a c e temperature and the o u t l e t temperature.  Around  the  mouth o f the i n l e t t h e r e i s an e x t e n s i v e marsh and the stream g i v e s o f f a number o f s m a l l channels, forming a d e l t a . Above the,marsh  a r e a the stream g r a d i e n t i s r e l a t i v e l y  shallow w i t h p o o l s separated by r i f f l e s e c t i o n s . of  The banks  the i n l e t stream are covered w i t h a heavy growth o f brush  over i t s e n t i r e l e n g t h , and t h e r e a r e f r e q u e n t l y p i l e s o f brush sunken i n p o o l s .  Mr. E.E. Moodie observed a sharp  d e c l i n e i n catch o f a d u l t c y p r i n i d s i n the l a k e a t the time spawning  f i s h appeared i n the stream, so i t may be assumed  t h a t most o f the c y p r i n i d s i n the l a k e u t i l i z e the stream for  spawning.  11  Fig.  3.  W o l f e L a k e and W o l f e  Creek.  W o l f e L a k e and W o l f e C r e e k S c a l e : ,, 3 0 0 meters, • - C o l l e c t i o n &. O b s e r v a t i o n Station x - A c r o c h e i l u s & Ptychocheilus Upper Limit in C r e e k  12 The o u t l e t o f Wolfe Lake, l i k e M i s s e z u l a Lake, has been b l o c k e d by an e a r t h dam.  The drop between,the l a k e  l e v e l and t h e l e v e l o f the stream below i s g r e a t e r i n Wolfe Lake, and, w h i l e the dam may n o t be a b a r r i e r t o the movement o f a d u l t f i s h ,  i t i s p r o b a b l y an e f f e c t i v e b a r r i e r to  upstream movement o f young c y p r i n i d s .  In a d d i t i o n , t h e  g r a d i e n t o f the o u t l e t stream i s s t e e p e r than t h a t o f the i n l e t , making i t l e s s p r o b a b l e t h a t young c y p r i n i d s would be a b l e t o r e t u r n t o the l a k e .  B. F i e l d C o l l e c t i o n s and O b s e r v a t i o n s Samples o f a d u l t f i s h were o b t a i n e d by g i l l n e t t i n g , b o t h from spawning  and non-spawning  S e i n i n g and rotenone, as w e l l as small-meshed were used i n c o l l e c t i n g j u v e n i l e  (overnight).  gillnets  specimens.  When sampling s t a n d i n g water, s e t s were used, l e f t  fish.  stationary  gillnet  i n most cases f o r a p e r i o d o f t e n hours  In f l o w i n g water,  the g i l l n e t s were allowed  t o d r i f t through the d e s i r e d a r e a and the f i s h immediately a f t e r w a r d .  removed  S e i n i n g was o f r e s t r i c t e d use i n  most areas s t u d i e d due t o the presence o f l o g s and b o u l d e r s on the bottom.  The most e f f e c t i v e c o l l e c t i o n s o f j u v e n i l e  f i s h were o b t a i n e d e i t h e r w i t h rotenone o r fine-mesh  gillnets.  G i l l n e t c o l l e c t i o n s were made d a i l y d u r i n g both the 1964 and 1965 f i e l d  seasons from M i s s e z u l a Lake.  All  13 h y b r i d s captured were saved  f o r morphological  analysis.  Most o f the A c r o c h e i l u s and P t y c h o c h e i l u s used i n the morphological 1964,  s t u d i e s were taken from M i s s e z u l a Lake i n  although c o l l e c t i o n s from a l l areas s t u d i e d were  compared w i t h  these.  F i s h which were r e t a i n e d a l i v e f o r l a t e r work were k e p t i n stryofoam c o o l e r c h e s t s f o r t r a n s p o r t to the  shore  o r l a b o r a t o r y , and f i s h which were t o be r e t a i n e d a l i v e f o r l o n g e r than a few days were t r e a t e d w i t h an i n j e c t i o n o f 1,000 streptomycin to prevent  intraperitoneal  u n i t s o f p e n i c i l l i n and 1,000  ( D i f c o P e n i c i l l i n - S t r e p t o m y c i n T.C.)  i n f e c t i o n i n net wounds.  F i s h t o be  micrograms i n order preserved  had t h e i r body c a v i t i e s opened on the r i g h t s i d e and were p r e s e r v e d i n 10% F o r m a l i n immediately  after  collection.  Samples o f gonads f o r h i s t o l o g i c a l s t u d i e s were p r e s e r v e d i n Carnoy's f i x a t i v e a c e t i c a c i d ) and  (3 p a r t s e t h a n o l  :1 p a r t g l a c i a l  refrigerated.  Groups o f f i s h i n spawning c o n d i t i o n were both  from above water and underwater.  observed  In M i s s e z u l a Lake  a s m a l l p i e r was b u i l t i n the o u t l e t a d j a c e n t to the area where f i s h were spawning.  O b s e r v a t i o n s were a l s o made  s u c c e s s f u l l y by d r i f t i n g a l o n g the shore o f the l a k e i n a boat,  the water b e i n g c l e a r enough f o r good v i s i o n t o a  depth o f 4.6  - 6.1  t o Wolfe Creek and  M.  Underwater o b s e r v a t i o n s were c o n f i n e d  the o u t l e t o f M i s s e z u l a Lake.  n e a r l y s t a t i o n a r y water o f the l a k e , i t was  In the  necessary  to  14 use an oxygen r e b r e a t h e r ,  s i n c e t h e n o i s e produced b y open  c i r c u i t d i v i n g equipment o r even b y b r e a t h i n g through a s n o r k e l tube f r i g h t e n e d the f i s h .  In Wolfe Creek, the  f l o w i n g water produced s u f f i c i e n t background n o i s e so t h a t the f i s h were n o t d i s t u r b e d b y t h e b r e a t h i n g n o i s e s caused by a s n o r k e l tube. e i t h e r recorded  Data from underwater o b s e r v a t i o n s were  i n p e n c i l on a sheet o f p o l y v i n y l p l a s t i c  w h i l e underwater o r recorded with o t h e r f i e l d notes  after  the d i v e .  C. Rearing  Experiments  W i l d a d u l t f i s h o f b o t h p a r e n t a l s p e c i e s as w e l l as h y b r i d s , when a v a i l a b l e , were used as p a r e n t s f o r e x p e r i m e n t a l l y r e a r e d young.  A l l b u t two o f t h e p a r e n t  f i s h were o b t a i n e d from M i s s e z u l a Lake.  A t o t a l o f 44  a r t i f i c i a l c r o s s e s were made d u r i n g t h e 1964 and 1965 seasons.  The types o f c r o s s e s and t h e i r f a t e a r e as  follows. During the 1964 season, 33 a r t i f i c i a l were made.  Of these,  16 c r o s s e s hatched  crosses  s u c c e s s f u l l y and  y i e l d e d young which c o u l d be used f o r m o r p h o l o g i c a l  studies.  They comprised 8 A c r o c h e i l u s x A c r o c h e i l u s c r o s s e s , 10 female A c r o c h e i l u s x male P t y c h o c h e i l u s r e c i p r o c a l s and one backcross  o f female A c r o c h e i l u s x male presumed Fi_ x  Ptychocheilus  parentage.  Two female P t y c h o c h e i l u s x  15 male Acrocheilus reciprocals died after 72 hours of development at 20°C, as did 6 Acrocheilus x Acrocheilus and 6 female Acrocheilus x male Ptychocheilus crosses. During the 1965 season, a t o t a l of 11 crosses were made.  Three of these produced young which were used for  morphological analysis.  These were 2 female Ptychocheilus  x male Acrocheilus hybrid reciprocals and 1 female Acrocheilus x male Ptychocheilus r e c i p r o c a l .  In addition,  young from 2 Ptychocheilus x Ptychocheilus crosses made by C . C . Lindsey were used.  One of these originated from Lac  l a Hache parents, which are a l l o p a t r i c from Acrocheilus. Three wild presumed F]_ hybrids with free gametes were taken in 1965.  Two of these were males,  with two Acrocheilus females. beyond 72 hours.  and each was crossed  None of the eggs survived  A single female presumed F i with free  eggs was crossed with both a male Acrocheilus and a male Ptychocheilus.  A l l but 200 of the r e s u l t i n g eggs were  dead within 60 minutes of f e r t i l i z a t i o n , and the remaining 200 a l l died after 24 hours of development at 1 8 ° C .  A  single cross of a female Richardsonius balteatus x male Acrocheilus yielded one offspring whose morphology was studied. Parent f i s h were taken i n overnight g i l l n e t sets near areas were aggregations of f i s h i n spawning condition or actual spawning had been observed.  The f i s h were  retained l i v e i n styrofoam cooler chests and brought to  16 shore.  The  eggs o f a given female were s t r i p p e d onto n y l o n  c r i n o l i n e screen i n a d r y s t a c k i n g d i s h i n l o t s o f from to  t h r e e hundred per d i s h .  In a l l cases, eggs from a s i n g l e  female were f e r t i l i z e d w i t h sperm from a t l e a s t e o n s p e c i f i e and one h e t e r o s p e c i f i c male. where p o s s i b l e , was After fertilization,  one  one  A l s o a g i v e n male,  c r o s s e d w i t h a female o f both s p e c i e s . the eggs o f each l o t were spread i n t o a  s i n g l e l a y e r on the c r i n o l i n e screen which was  then  c a r e f u l l y immersed i n water i n a second s t a c k i n g d i s h .  The  eggs were always s h i e l d e d from d i r e c t s u n l i g h t .  the  spawning s e a s o n | i n 1964, Vancouver to be hatched of  During  the eggs were t r a n s p o r t e d by a i r to i n the l a b o r a t o r y .  In 1965,  most  the eggs were kept i n screen enclosed boxes i n the l a k e  for hatching.  The eggs were h e l d i n 7 X 9 x 10 em.  made of n y l o n c r i n o l i n e and  l i n e d with nylon  baskets  chiffon.  These were suspended i n the water by means o f a wooden frame i n s i d e the r e a r i n g box.  A f t e r the eggs hatched,  f i s h were r e t a i n e d i n the c r i n o l i n e b a s k e t s months d u r i n g the 1964  season,  f o r about  or t r a n s f e r r e d to 20  a q u a r i a two weeks a f t e r h a t c h i n g d u r i n g the 1965  to i n s u f f i c i e n t oxygen i n the water i n the  baskets.  liter  apparently  crinoline  S i n c e f i n e mesh l i n e r s are r e q u i r e d i n order t o  r e t a i n the newly hatched box  two  season.  Much o f the p r e - h a t c h i n g m o r t a l i t y was due  the young  f i s h , heavy a e r a t i o n i n the r e a r i n g  o r a e r a t i o n o f each c r i n o l i n e b a s k e t  i s necessary.  The  c i r c u l a t i o n w i t h i n the screen e n c l o s e d r e a r i n g boxes h e l d i n  17  M i s s e z u l a Lake d u r i n g the 1965 season was n o t s u f f i c i e n t avoid m o r t a l i t y .  In fact,  the m o r t a l i t y t o eggs h e l d i n t h e s e  boxes was h e a v i e r than t h a t o f eggs h a t c h e d i n t h e After hatching,  to  laboratory.  the young f i s h r e q u i r e d about 5  days to absorb t h e i r r e m a i n i n g y o l k  (at 1 8 - 2 0 ° C . ) .  were then fed a m i x t u r e o f b r i n e shrimp (Artemia)  They nauplii  and i n f u s o r i a c u l t u r e i n t h e case o f t h o s e r e a r e d i n the Vancouver l a b o r a t o r y ,  o r the i n f u s o r i a c u l t u r e and n a t u r a l l y  o c c u r r i n g p l a n k t o n ( l a r g e l y Diaptomus) f o r f i s h r e a r e d the f i e l d .  As t h e f i s h grew, the d i e t was  with dry t r o p i c a l fish increasing proportions, on t h i s food a f t e r  in  supplemented  food ( " T e t r a - M i n Growth Food") i n and the f i s h were m a i n t a i n e d  t h e y had reached 2-2% months o f  entirely  age.  A l l e x p e r i m e n t a l l y r e a r e d f i s h were k e p t a l i v e u n t i l the f i n s were f u l l y formed, and when p o s s i b l e ,  until  the s c a l e s were formed.  D. M o r p h o l o g i c a l  Studies  B o t h w i l d and e x p e r i m e n t a l l y r e a r e d f i s h used f o r m o r p h o l o g i c a l s t u d i e s .  were  F i e l d i d e n t i f i c a t i o n of  the p a r e n t s p e c i e s and h y b r i d s i s n o t d i f f i c u l t  and n o t e s  r e g a r d i n g c o l o r , p r e s e n c e o f spawning t u b u r c l e s and g e n e r a l appearance c o u l d be made r e a d i l y on u n c a p t u r e d f i s h w h i l e d i v i n g o r o b s e r v i n g from the I n the case o f s m a l l f i s h  either  surface. (20 mm. o r s m a l l e r )  it  18 was  n e c e s s a r y t o s t a i n the specimens w i t h a l i z a r i n e  red-s  dye  i n order to count the s c a l e s and pharyngeal t e e t h .  Measurements on these s m a l l f i s h were made u s i n g a stereomicroscope equipped w i t h a graduated mechanical and a c r o s s h a i r i n the o c u l a r .  stage  L a r g e r f i s h were measured  w i t h d i a l c a l i p e r s to the n e a r e s t 0.1  mm.  Sample s i z e s o f f i s h f o r each m o r p h o l o g i c a l c h a r a c t e r are i n d i c a t e d on the f i g u r e or t a b l e r e l a t i n g that character.  A t o t a l o f 2,622 a d u l t c y p r i n i d s were  c o l l e c t e d d u r i n g the 1964 of  to  and 1965  seasons.  Only a p o r t i o n  these were p r e s e r v e d and r e t u r n e d t o Vancouver. Most c h a r a c t e r s are d e f i n e d i n accordance  Hubbs and L a g l e r (1947). 1.  with  The e x c e p t i o n s are given below.  L a t e r a l s c a l e rows are the number o f d i a g o n a l s c a l e rows c r o s s i n g the line.  The a n t e r i o r m o s t s c a l e  lateral  row  counted had the l a t e r a l l i n e s c a l e i n c o n t a c t w i t h the p e c t o r a l g i r l d e . p o s t e r i o r m o s t row  counted  with l a s t l a t e r a l  l i n e s c a l e on  terminated the  base o f the caudal f i n . T h i s count found t o be e a s i e r than was pored l a t e r a l - l i n e  The  was  a count o f  s c a l e s on A c r o c h e i l u s ,  on which the s c a l e s are imbedded, and  on  very small f i s h . 2.  Gut l e n g t h measurements are not d e t a i l e d  19 i n Hubbs and L a g l e r (1947).  The gut  was removed b y s e v e r i n g a t the  transverse  septum a n t e r i o r l y and c u t t i n g the body w a l l around the anus p o s t e r i o r l y . Arbitrarily,  the stomach was d e f i n e d  the a n t e r i o r  l o n g i t u d i n a l segment,  as  and  a l l p o r t i o n s from the f i r s t  curvature  t o the anus were d e s i g n a t e d  as  E. Electrophoretic  intestine.  Studies  E l e c t r o p h o r e t i c a n a l y s i s o f muscle p r o t e i n s was c a r r i e d o u t b y D r . T s u y u k i o f the F i s h e r i e s Research B o a r d o f Canada T e c h n o l o g i c a l S t a t i o n i n V a n c o u v e r . W i l d f i s h o f b o t h p a r e n t s p e c i e s and t h e h y b r i d s were c o l l e c t e d i n the f i e l d  for t h i s purpose.  The f i s h  were r e t a i n e d a l i v e u n t i l t h e sample was t a k e n .  The sample  c o n s i s t e d o f a f i l l e t c u t from the l o n g i t u d i n a l muscle mass which was l a b e l e d and f r o z e n on CO2 i c e i m m e d i a t e l y  after  b e i n g removed from t h e f i s h .  frozen  The samples remained  u n t i l a n a l y s i s , a t w h i c h time the s k i n and d a r k muscle was removed and d i s c a r d e d .  The r e m a i n i n g w h i t e muscle was  b r o k e n up i n w a t e r b y means o f a b l e n d e r , filtrate  and t h e  clear  from t h i s m i x t u r e was used t o make t h e  electrophoretic  run.  A f t e r d e v e l o p i n g , the  p a t t e r n s were photographed  and the photographs  resulting used i n  20 making comparisons.  F. C y t o l o g i c a l  Studies  Chromosome number and morphology were determined f o r b o t h p a r e n t s p e c i e s and f o r e x p e r i m e n t a l l y r e a r e d h y b r i d s representing  both r e c i p r o c a l  crosses.  Two techniques were used s u c c e s s f u l l y t o o b t a i n chromosome p r e p a r a t i o n s .  The f i r s t o f these was an  a d a p t a t i o n o f f i s h t i s s u e c u l t u r i n g techniques as r e p o r t e d by Parker  (1950), Wolfe and Dunbar (1956), Wolf e e t a l (I960),  Townsley e t a l (1963) and Roberts  (1964).  Primary  cultures  o f t e s t i s were o b t a i n e d from b o t h p a r e n t s p e c i e s by a s e p t i c a l l y removing the t e s t e s , mincing them i n t o p i e c e s 1  mm.  or l e s s i n diameter i n s t e r i l e f i s h Ringer's s o l u t i o n , and explanting flask  from f i v e t o f i f t e e n p i e c e s i n t o a 30 ml. c u l t u r e  (Falcon P l a s t i c s s t e r i l e ,  culture  flask),  d i s p o s a b l e 30 ml t i s s u e  2.ml. o f B.B.L. t i s s u e c u l t u r e medium 199,  Hank's base, p l u s 10% human serum was added t o the f l a s k and  the c u l t u r e incubated a t 18°C f o r seven days.  the  incubation  period,  2 ml o f f r e s h medium and 0.01 ml. o f  0.1% c o l c h i c i n e were added t o t h e c u l t u r e . additional treated  incubation  After  period  A f t e r an  o f 36 hours, the t i s s u e s were  f o r 10 minutes w i t h a h y p o t o n i c s o l u t i o n  of 1 p a r t medium t o 6 p a r t s d i s t i l l e d water.  consisting  F i x i n g and  s t a i n i n g were done s i m u l t a n e o u s l y i n 2% l a c t i c - a c e t i c  21 o r c e i n ( N a t u r a l o r c e i n s 2 gm, A c e t i c a c i d : 45 ml, l a t i c a c i d 55 m l ) . suspension  Smears were prepared  immediately by s c r a p i n g a  o f c e l l s from the p i e c e o f t i s s u e i n t o a drop  o f s t a i n on a cleaned  slide.  A c o v e r s l i p (22 mm.square #0)  was p l a c e d over the drop o f s t a i n ,  a i r bubbles excluded by  l i g h t p r e s s u r e w i t h a d i s s e c t i n g needle,  and t h e p r e p a r a t i o n  squashed b y t h e a p p l i c a t i o n o f the author's #4 rubber stopper p l a c e d on t h e c o v e r s l i p .  f u l l weight on a Examination  and photography were done u s i n g a Z e i s s photomicroscope equipped f o r phase c o n t r a s t .  A l l photos were made u s i n g  the o i l immersion o b j e c t i v e . Due t o t h e extremely s m a l l s i z e o f t h e gonad i n the r e a r e d h y b r i d s ,  i t was n o t p o s s i b l e t o s t a r t a c u l t u r e .  For these specimens, a technique (per. comm.) was employed.  developed by J.D. McPhail  T h i s i n v o l v e d the use o f  e p i t h e l i a l c e l l s from the l a m e l l a e o f t h e f o u r t h g i l l The  fish  (about  arch.  2 cm.in length) were t r e a t e d with a  subcutaneous i n j e c t i o n o f 0.05 ml o f 0.01% c o l c h i c i n e i n f i s h Ringer's 1 hour.  and h e l d a l i v e i n h e a v i l y aerated water f o r  The f o u r t h g i l l  arch was then removed i n t a c t and  p l a c e d i n d i s t i l l e d water f o r 30 minutes and i n l a c t i c a c e t i c o r c e i n f o r an a d d i t i o n a l 15 minutes. suspension  was then scraped  A cell  from the t e r m i n a l p o r t i o n s  o f the l a m e l l a e i n t o a drop o f s t a i n on a s l i d e and the smear made as d e s c r i b e d p r e v i o u s l y . Chromosome morphology was compared between t h e  22 p a r e n t s p e c i e s and h y b r i d s b y means o f photographs. karyotype  A  f o r each p a r e n t s p e c i e s , showing t h e probable  p a i r i n g o f the chromosomes, was c o n s t r u c t e d by a r r a n g i n g the chromosomes a c c o r d i n g t o total': l e n g t h , position,  centromere  and t h e presence o f c o n s t r i c t i o n s .  Comparisons  were then made between t h e p a r e n t s p e c i e s , and h y b r i d s . D i p l o i d number and arm number c o u l d be determined w i t h more c e r t a i n t y than the morphology o f s p e c i f i c chromosomes w i t h the r e s t r i c t e d amount o f m a t e r i a l which was a v a i l a b l e . Some o f the chromosomes were d i s t i n c t i v e because o f t h e i r s i z e o r morphology; however,.and these c o u l d be seen i n a l l the preparations.  23 RESULTS A. Morphology and E l e c t r o p h o r e t i c S t u d i e s The m o r p h o l o g i c a l  d i f f e r e n c e s between A c r o c h e i l u s  and P t y c h o c h e i l u s may be summarized as f p l l o w s : 1.  S c a l e counts:  higher i n Acrocheilus  P t y c h o c h e i l u s , with l i t t l e o v e r l a p 2.  Pharyngeal t e e t h : row  (Fig. 4 ) .  A c r o c h e i l u s has a s i n g l e  o f t e e t h on each pharyngeal  or 5 t e e t h  than  (0,4-5,0).  arch w i t h 4  P t y c h o c h e i l u s has 2  rows o f t e e t h p e r arch, the most common count b e i n g 2,5-4,2, with r a r e v a r i a n t s showing 1,5-4,2; 3.  2,5-4,3 o r 3,5-4,2.  Intestinal tract: Acrocheilus  complexly c o i l e d i n  ( F i g . 5 ) , a simple S-shaped tube  in Ptychocheilus 4.  (Table I ) .  Peritoneum c o l o r :  (Fig. 5). black i n Acrocheilus,  s i l v e r y i n Ptychocheilus. 5.  Lower jaw: has a k e r a t i n i z e d , c h i s e l - l i k e a n t e r i o r margin i n A c r o c h e i l u s , and i s rounded, w i t h no s p e c i a l i z a t i o n i n P t y c h o c h e i l u s ( F i g . 6) .  6.  Body p r o p o r t i o n s :  A c r o c h e i l u s has a narrower  caudal peduncle, s h o r t e r jaws and s h o r t e r snout than P t y c h o c h e i l u s  (Fig. 7).  24  Fig.  4  0  L a t e r a l and v e n t r a l p r o f i l e s o f A c r o c h e i l u s , P t y c h o c h e i l u s and w i l d i n t e r m e d i a t e s . 1. P t y c h o c h e i l u s oregonensis, M i s s e z u l a Lake. 2 Presumed F]_ x P t y c h o c h e i l u s , M i s s e z u l a Lake. 3. F]_ Hybrid, M i s s e z u l a Lake. 4 . Presumed F]_ x A c r o c h e i l u s , Wolfe Lake. 5„ A c r o c h e i l u s a l u t a c e u s , M i s s e z u l a Lake. 0  25  Fig.  5.  Numbers o f d i a g o n a l s c a l e rows i n w i l d and reared A c r o c h e i l u s , P t y c h o c h e i l u s and I n t e r m e d i a t e s . H o r i z o n t a l l i n e = range; v e r t i c a l l i n e = mean; s o l i d r e c t a n g l e = 2 standard d e v i a t i o n s .  Wild  .N=48  Acrocheilus Reared  .N=3  Acrocheilus Wild P r e s u m e d _  1  RjX A c r o c h e i l u s  N=2  Wild R,  .N=40  Hybrids R e a r e d F. Hybrids  .N=23  1  Wild P r e s u m e d F  1  N=2  X  Ptychocheilus Wild S y m p a t r i c Ptychocheilus  ,N=49  Wild A l l o p a t r i c  ,N=20  Ptychocheilus  —I—  60  70  80  90  N u m b e r of D i a g o n a l S c a l e R o w s (Solid R e c t a n g l e s = 2 S t a n d a r d Deviations)  100  26  F i g . 6.  Diagrams o f gut c o i l i n g p a t t e r n s f o r A c r o c h e i l u s , P t y c h o c h e i l u s , w i l d i n t e r m e d i a t e s , and j u v e n i l e Acrocheilus.  27  Fig.  7.  R e l a t i o n s h i p s o f v a r i o u s body p r o p o r t i o n s t o standard length i n w i l d A c r o c h e i l u s , P t y c h o c h e i l u s and I n t e r m e d i a t e s .  o Rj Hybrids • Acrocheilus A Sympatric Ptychocheilus Allopatric Ptychocheilus • F X Ptychocheilus A  a fij X Acrocheilus  • • •• ••  ^  *4A*  A A ^ A A ^ O  °  >  ^  T  <A  100 150 Standard Length m m .  50  2 °° °  A  250  200  .10• *  * A  AA  *A4  A A*A  A  A  c  °  QOOS  o  OO  . 8°  tr.0  °b .05^  °o ° °oop o o O O o  o oc  c <uca  _i *-*  oJ  3 C  100 150 Standard Length mm.  50  .50i A  A  A  A  O i . »A.  A  A , AO,  A^f  AA O  oooo  <5>o O  250  200  a> oo  fl) .25  50  100 150 Standard Length m m .  200  250  Table I  Frequency of pharyngeal  Acrocheilus  Number o f o u t e r row pharyngeal teeth Left Arch  Right Arch  0 - 0  W i l d Reared 56  Fl  tooth counts  in wild  H Y B R I D S  Female Acrocheilus x male Ptychocheilus reared  Wild  and r e a r e d p a r e n t a l  B A C K C R O S S  Female Ptychocheilus x male Acrocheilus Reared  Wolfe Lake Presumed l * Acrocheilus Wild F  Missezula Lake Presumed F x Ptychocheilus Wild X  18  s p e c i e s and h y b r i d  Ptychocheilus  E S  Presumed male F^ x Ptychocheilus X female Acrocheilus Reared  Sympatric Wild  Reared  Allopatric Wild  59  35  1  40  0 - 1  1  3  1 - 0  1  2  1 - 1  14  4  1 - 2  2  5  2 - 1  4  7  1  2 - 2  6  26  3  1  2  15 1 1 1  2  2 - 3  2  3 - 2  2  1  1  1  3 - 3 Sample  18  sizes  56  18  28  42  4  2  2  63  63  37  20  29 The body p r o p o r t i o n s o f t h e p a r e n t  species  are  s i m i l a r i n s m a l l f i s h and d i v e r g e w i t h i n c r e a s i n g s i z e . The o n l y r e l i a b l e c h a r a c t e r s  for d i s t i n g u i s h i n g parent  s p e c i e s and i n t e r m e d i a t e s below one y e a r i n age a r e  the  pharyngeal tooth counts,  are  formed, apparent  and the p e r i t o n a l c o l o r .  The c h i s e l l i p i s not  i n A c r o c h e i l u s u n t i l the end o f the f i r s t summer  a t the e a r l i e s t , 4-6 cm.  the s c a l e c o u n t s i f s c a l e s  and i s rounded i n o u t l i n e i n f i s h b e l o w  B e f o r e the c h i s e l has appeared,  A c r o c h e i l u s tends  t o have a s l i g h t l y s u b t e r m i n a l mouth, w h i c h a i d s i n d i s t i n g u i s h i n g i t from the s q u a w f i s h , b u t t h i s may be masked by deformation i n preserved  fish.  The i n t e r m e d i a t e forms c o l l e c t e d i n t h e w i l d fall  into three categories.  f  B y f a r t h e most common form  agrees c o m p l e t e l y w i t h t h e d e s c r i p t i o n s by Snyder Hubbs and S c h u l t z (1931) and P a t t e n  (1960).  (1906),  The f i g u r e s  and t a b l e s r e f e r r e d t o i n the d i s c u s s i o n o f p a r e n t  species  morphology g i v e t h e v a l u e s o f a l l t h r e e h y b r i d m o r p h o l o g i e s f o r each  character. One o f the s t r i k i n g f e a t u r e s  o f the morphology o f  the common i n t e r m e d i a t e t y p e i s the apparent dominance o f some s q u a w f i s h c h a r a c t e r s .  No t r a c e o f a k e r a t i n i z e d  edge i s t o b e found on the l o w e r jaw, a l t h o u g h i n most other respects  the mouth p r o f i l e i s i n t e r m e d i a t e  (Fig. 6 ) .  The i n t e s t i n e shows none o f t h e c o i l i n g t o be seen i n Acrocheilus,  a l t h o u g h the a n t e r i o r l o o p o f the  intestine  30 i s longer than i n P t y c h o c h e i l u s .  In some cases,  inflections  are p r e s e n t a n t e r i o r l y which are r e m i n i s c e n t o f the morphology i n young-of-the-year A c r o c h e i l u s .  The  gut pharyngeal  t e e t h o f w i l d i n t e r m e d i a t e s are most commonly i d e n t i c a l i n count to P t y c h o c h e i l u s , although  a few  i n d i v i d u a l s show a  1,5-4,1 count, which might a r b i t r a r i l y be i n t e r m e d i a t e between 0,5-4,0 and  considered  2,5-4,2.  Most other c h a r a c t e r s l i e between mean v a l u e s o f the p a r e n t s p e c i e s .  The peritoneum,  (data not shown) v a r i e s  from s i l v e r y w i t h d i f f u s e melanophores to almost b l a c k , a s i l v e r y sheen.  Most commonly, i t i s grey.  The o t h e r two  i n t e r m e d i a t e types c o l l e c t e d were  r e p r e s e n t e d by f o u r specimens, two Lake, and  two  from Wolfe Lake.  73 and  taken  from M i s s e z u l a  The M i s s e z u l a Lake specimens  d i f f e r e d from t y p i c a l i n t e r m e d i a t e s counts,  with  i n having  lower s c a l e  74 d i a g o n a l rows, a much s h o r t e r a n t e r i o r  loop o f the gut, and a s t r a i g h t r a t h e r than decurved mouth (Fig. 6). like.  They tended t h e r e f o r e , to be more P t y c h o c h e i l u s -  The Wolfe Lake specimens were, on the other hand,  more A c r o c h e i l u s - l i k e . the gut was and  The> s c a l e counts were 83 and  strongly inflected,  the lower jaw had  The mouth was  ( F i g . 5), b u t not  85,  coiled,  a rounded, k e r a t i n i z e d margin  (Fig. 6 ) .  s h a r p l y d e f l e c t e d , as i n A c r o c h e i l u s , r a t h e r  than decurved, as i n the t y p i c a l The p a t t e r n observed the two p a r e n t forms, and  intermediates.  i n nature,  then,  c o n s i s t s of  three r e a d i l y d i s t i n g u i s h a b l e  31 intermediate  forms, two  o f which are extremely r a r e .  Since  the body p r o p o r t i o n s are not d i s t i n c t i v e i n youhg-of-theyear f i s h , was  a n a l y s i s o f the morphology o f reared specimens  l i m i t e d f o r the most p a r t to pharyngeal t o o t h counts  s c a l e counts,  and  both o f which are d i s t i n c t i v e i n the n a t u r a l i  populations. Reared A c r o c h e i l u s a l l showed 0,5-4,0 pharyngeal tooth count, as do the w i l d f i s h  (Table I ) .  The mean s c a l e  count o f the o n l y three r e a r e d A c r o c h e i l u s which s u r v i v e d l o n g enough f o r s c a l e s to be  formed was  85.66 (range 83-86)  as compared to a mean o f 85.83, w i t h a range o f 80-94 f o r wild Acrocheilus s c a l e s had:formed  (Fig. 4). (about  k e r a t i n i z e d margin was T h i s was  In the three f i s h i n which  3 cm. body length)  beginning  a rounded,  to appear on the lower  jaw.  c l o s e l y comparable t o t h a t observed i n w i l d young-  o f - t h e - y e a r A c r o c h e i l u s i n M i s s e z u l a Lake d u r i n g the same month (October).  The  diagrammed f o r 38 mm. The  gut showed i n f l e c t i o n s much l i k e wild fish  ( F i g . 5).  r e a r e d squawfish o r i g i n a t e d from Wolfe Lake  and Lac l a Hache parents, constant  those  temperatures.  and were r e a r e d a t t h r e e d i f f e r e n t None was  r e a r e d to a l a r g e enough  s i z e f o r s c a l e s to have formed, but pharyngeal t o o t h counts were r e a d i l y made on a l l (Table I ) .  The  counts are  the  same as those observed i n w i l d Wolfe Lake and M i s s e z u l a Lake populations. i n Table I )  The Lac l a Hache specimens are a l l o p a t r i c ,  (shown s e p a r a t e l y  so t h a t t h e r e i s no  possibility  32 o f i n t r o g r e s s i o n o f chiselmouth  genes Into t h i s p o p u l a t i o n .  Table I shows the pharyngeal  t o o t h counts o f r e a r e d  F^ h y b r i d s i n comparison w i t h w i l d i n t e r m e d i a t e s and the parent species. separately.  The two r e c i p r o c a l c r o s s e s a r e shown  The d a t a f o r t h e female A c r o c h e i l u s r e c i p r o c a l  r e p r e s e n t t h e pooled r e s u l t s o f a l l c r o s s e s o f t h i s type which were made.  Only f i v e o f f s p r i n g o f the female P t y c h o c h e i l u s  reciprocal,  representing a s i n g l e cross, survived.  i s a d i f f e r e n c e i n the modal pharyngeal  There  t o o t h count o f r e a r e d  F^s o f t h e female A c r o c h e i l u s r e c i p r o c a l and t h e w i l d intermediates. l a t t e r 2-2.  The former a r e predominantly  1-1 and t h e  T h i s may,be due i n p a r t , t o t h e s m a l l s i z e o f  many o f the r e a r e d F^s, which d i e d a t about 1.5 cm., b e f o r e s c a l e s were formed.  The f i v e female P t y c h o c h e i l u s  r e c i p r o c a l s which were hatched about 3 cm., and, o f those, seen i n the w i l d  s u c c e s s f u l l y were reared t o  three showed a 2-2 count as i s  intermediates.  S c a l e counts o f 18 female A c r o c h e i l u s r e c i p r o c a l s had  a mean o f 79.77, compared t o a mean o f 80.58 f o r t h e  w i l d intermediates.  This d i f f e r e n c e i s not s i g n i f i c a n t a t  the 95% c o n f i d e n c e l e v e l  (Fig. 4 ) .  The f i v e female  P t y c h o c h e i l u s r e c i p r o c a l s had a mean s c a l e count o f 80.4, almost i d e n t i c a l t o the w i l d  intermediates.  Reared specimens, then correspond  closely i n their  morphology t o w i l d specimens presumed t o be o f t h e same parentage.  33  In o r d e r to compare the muscle  electropherograms  o f the p a r e n t a l s p e c i e s and h y b r i d s , the bands p r e s e n t were d e s i g n a t e d w i t h l e t t e r s , b e g i n n i n g w i t h A a t the cathode s i d e o f the g e l . p r e s e n t was hybrids  The  f r e q u e n c y o f occurrence o f each band  then determined  (Table I I ) .  f o r the p a r e n t a l s p e c i e s and  Photographs o f the  electropherograms  o f a l l u s a b l e samples are g i v e n i n F i g s . 8,  9 and  10.  A number o f the samples which o r i g i n a t e d from L o m e and V i d e t t e Lakes and the Okanogan R i v e r were h e l d i n f r o z e n s t o r a g e f o r about three months b e f o r e b e i n g run.  The  p a t t e r n s o b t a i n e d from these samples were anomalous, i n d i c a t i n g t h a t decomposition No  had o c c u r r e d d u r i n g s t o r a g e .  c o n s i s t e n t d i f f e r e n c e s c o u l d be found between  A c r o c h e i l u s and P t y c h o c h e i l u s , electropherograms. II;  F i g s . 8,  9 and  (Table  1Q).  B. R e l a t i v e Abundance o f Hybrids The q u a n t i t a t i v e composition o f the c a t c h by s p e c i e s was  recorded f o r a l l samples of a d u l t f i s h  each study area.  Samples of j u v e n i l e s were a l s o analyzed  f o r s p e c i e s composition, b u t are more r e s t r i c t e d numbers o f f i s h c o l l e c t e d and was  from  i n the  i n the area sampled.  Also, i t  mentioned p r e v i o u s l y t h a t i d e n t i f i c a t i o n o f h y b r i d s i s  u n c e r t a i n i n young-of-the-year  fish,  so t h a t estimates o f  h y b r i d abundance i n j u v e n i l e s i s p r o b a b l y not as  reliable  34  Fig„ 8.  Myogen electropherograms o f A c r o c h e i l u s  alutaceus.  Acrocheilus alutaceus Missezula Lake  15cm.  • Wolfe Lake  Okanogan River  Vidette Lake  35  Fig. 9.  Myocren electropherograms o f P t y c h o c h e i l u s oregonensis.  Ptychocheilus oregonensis  Vidette Lake  36  F i g . 10.  Myogen e l e c t r o p h e r o g r a m s o f w i l d  intermediates.  Wild Intermediates  P r e s u m e d Ej X A c r o c h e i l u s Wolfe L a k e  Table I I  Frequency o f occurrence of bands i n myogen electropherograms o f w i l d p a r e n t a l s p e c i e s and h y b r i d s .  L E FT  RIG  A  B  C  D  E  F  M i s s e z u l a Lake Acrocheilus Ptychocheilus Fj_ Hybrids  2 8 1  2 6  2 8 1  2 5  2 8 1  2 8 1  Wolfe Lake Acrocheilus Ptychocheilus Fj_ H y b r i d s F]_ x A c r o c h e i l u s  . 1 1 2 none sampled 1 1 . 1 1 1  2  1  1  1 1  1 . 1  Lorne Lake Ptychocheilus  2  2  2  2  2  V i d e t t e Lake Acrocheilus Ptychocheilus  2 2  2  2 2  2  2 2  Okanogan R i v e r Acrocheilus  2  2  2  Fraser River Ptychocheilus  1  .1  Pooled R e s u l t s Acrocheilus Ptychocheilus F]_ Hybrids Fj_ x A c r o c h e i l u s  8 13 2 1  6 9 1  8 13 .1 1  ——  6 9  ' • --  G  -  -  origin  -  H  I  2 8 1  2 8 1  2  -  1 1  -  2 1 1  H T J  K 2 8 1 2  -  1 1  2  2  2  2  2  2 2  2 2  2 2  2  2  2  2  2  2  1  1  1  1  8 12 2 1  7 9 2  8 13 2 1  8 13 2 1  6 3  .1  1  1 6 11 2 1  to  38 as are the estimates  for adult  M i s s e z u l a Lake had for adult hybrids,  14.58  fish.  the h i g h e s t r e l a t i v e abundance  per cent r e l a t i v e to A c r o c h e i l u s .  In Wolfe Lake, h y b r i d s c o n s t i t u t e d 1.31 to the A c r o c h e i l u s .  The  per cent  relative  samples on which these f i g u r e s are  based i n c l u d e a t o t a l of 110  A c r o c h e i l u s and h y b r i d s c o l l e c t e d  i n d a i l y samples over a t h r e e month p e r i o d i n M i s s e z u l a and  232 A c r o c h e i l u s and h y b r i d s  Lake  c o l l e c t e d i n d a i l y samples  over a one month p e r i o d i n Wolfe Lake  (Table I I I ) .  A s i n g l e sample o f j u v e n i l e s from the Okanogan R i v e r , near Brewster, Washington contained hybrids  13.2  r e l a t i v e to A c r o c h e i l u s j u v e n i l e s .  No  were taken from the Okanogan R i v e r , although 70 were c o l l e c t e d . 11 h y b r i d s  and  Patten  4,260 A c r o c h e i l u s  Two 1965  contained  incidence  obtained  of  percent  f o r any h y b r i d  i s the  locality.  rotenone c o l l e c t i o n s made i n Wolfe Lake i n one  year o l d c y p r i n i d s .  At t h i s stage  p o s i t i v e i d e n t i f i c a t i o n o f the h y b r i d s was pooled  Acrocheilus  from the Yakima R i v e r .  the r e l a t i v e abundance o f 0.3  lowest f i g u r e which was  adult hybrids  (1960) r e p o r t s the c o l l e c t i o n o f  Although he says t h a t t h i s i s a h i g h hybridization,  percent  abundance o f h y b r i d s  f o r the two  p e r cent r e l a t i v e to A c r o c h e i l u s . o f h y b r i d s between t h i s sample and  The  possible,  samples was  and 3.45  decrease i n abundance  the a d u l t f i s h does not  n e c e s s a r i l y suggest s e l e c t i o n a g a i n s t h y b r i d s .  It  was  mentioned p r e v i o u s l y t h a t the samples o f j u v e n i l e s were  Table I I I  S p e c i e s c o m p o s i t i o n o f p o o l e d c a t c h from M i s s e z u l a Lake and Wolfe Lake M i s s e z u l a Lake, 1965 Adult Fish Number  Percent  246  19.63  Acrocheilus  96  7.66  Ptychocheilus x Acrocheilus Hybrids  14  1.12  893  71.27  4  0.32  P tychoche i l u s  Mylocheilus Mylocheilus x Ptychocheilus Hybrids Richardsonius  occurs but not counted  Total  1,253  Ptychocheilus Acrocheilus  x '  Ptychocheilus Acrocheilus  x  /  A  c  r  p  /  p  t  y  c  c  h  h  o  c  h  e  i  l  u  s  ' Number occurs b u t not counted 229  Percent  Number  Percent  57  9.27  98.69  58  9.43  1.31  2  0.33  373  60.65  occurs b u t not counted may not occur  may n o t occur  occurs b u t not counted 100.00 14.58  e  Wolfe Lake, 1965 Wolfe Lake, 1965 Juveniles Adult Fish ( E . E . M o o d i e , per. comm.)  5.69  232  100.00 1.31  125  20.33  615  100.02 3.45 3.51  40 more r e s t r i c t e d b o t h i n time p e r i o d and area covered. A l s o , the j u v e n i l e and a d u l t samples r e p r e s e n t d i f f e r e n t year c l a s s e s , and the r e l a t i v e abundance would n o t be comparable u n l e s s i t c o u l d be shown t h a t h y b r i d s a r e produced a t t h e same r a t e every year, and t h a t environmental  factors  c a u s i n g m o r t a l i t y had operated a t the same i n t e n s i t y over the span o f years s e p a r a t i n g the a d u l t s sampled from the juveniles.  G. F e r t i l i t y and V i a b i l i t y D u r i n g the spawning season i n 1964, a l l eggs c o l l e c t e d were r e t u r n e d t o Vancouver f o r h a t c h i n g .  Eggs  were taken i n l o t s o f about 300 each, and s u r v i v a l t o h a t c h i n g was h i g h l y v a r i a b l e .  In g e n e r a l , s u r v i v a l was  more dependent upon the i n d i v i d u a l female which was the source o f eggs than i t was on the c r o s s which was made. One female chiselmouth i n p a r t i c u l a r y i e l d e d 90 p e r cent h a t c h i n g success i n c r o s s e s both w i t h male chiselmouth and male squawfish. S e v e r a l l o t s o f eggs from b o t h chiselmouth and squawfish, r e p r e s e n t i n g both i n t r a - s p e c i e s c r o s s e s and h y b r i d c r o s s e s , were-observed d u r i n g t h e f i r s t f o u r days o f development. 20°C.  In a l l cases, r e a r i n g temperature  A t t h i s temperature,  for a l l crosses.  development appeared  was  normal  By the b e g i n n i n g o f the t h i r d day, 15  41 somites were v i s i b l e , had c l o s e d .  and by 84 hours  (3h days) the b l a s t o p o r e  In o t h e r l o t s which were r e a r e d through to  h a t c h i n g , eye pigmentation was and h a t c h i n g was  apparent by the t e n t h day,  complete by 14 - 17 days.  Hybrids w i t h mature gametes were i n f r e q u e n t . During the 1964  season, a s i n g l e male was  collected.  This  i n d i v i d u a l d i f f e r e d i n some r e s p e c t s from a t y p i c a l h y b r i d morphology, and was  the male p a r e n t o f the b a c k c r o s s o f f -  spring discussed previously.  Three t y p i c a l hybrids,, w i t h  f r e e gametes, two males and one female, were c o l l e c t e d d u r i n g the 1965  season.  Each o f the two males was  f e r t i l i z e b o t h chiselmouth and squawfish eggs. ly,  the m i l t from these males was  used to  Qualitative-  t r a n s l u c e n t , r a t h e r than  the opaque white t y p i c a l f o r p a r e n t s p e c i e s males, h a v i n g a "watery"  appearance.  In both cases, about 40%  (range  30-70%) o f the eggs showed malformed b l a s t o d i s c s a f t e r 6 hours i n c u b a t i o n a t 18°C.  A f t e r 24 hours i n c u b a t i o n ,  50% -  70% o f the eggs f e r t i l i z e d by the h y b r i d males were dead. The remaining l i v e embryos appeared normal, b u t a l l d i e d w i t h i n f o u r days o f f e r t i l i z a t i o n .  I t should be noted,  however, t h a t a t t h i s time n e a r l y a l l  the eggs o f i n t r a -  species crosses being reared also died. o f the f i r s t  While the m o r t a l i t y  24 hours cannot be e x p l a i n e d by environmental  causes, the l a t e r m o r t a l i t y a t 3-4 days was p r o b a b l y due t o i n s u f f i c i e n t oxygen i n the water, eggs b e i n g r e a r e d a t t h i s time.  and a f f e c t e d a l l the  42 The female h y b r i d y i e l d e d e i g h t l o t s o f about 200 a p p a r e n t l y normal eggs each.  Four o f these l o t s were  f e r t i l i z e d w i t h chiselmouth sperm and four w i t h squawfish sperm.  A l l b u t one o f these l o t s began t o s w e l l and t u r n  c l e a r on immersion i n water a f t e r f e r t i l i z a t i o n , o b v i o u s l y dead a f t e r 10 minutes.  and were  The s i n g l e l o t which  d i d n o t behave i n t h i s way had about 50% o b v i o u s l y dead eggs a f t e r 6 hours and 100% m o r t a l i t y i n 24 h o u r s .  Although  s w e l l i n g d i d not f o l l o w immersion as r a p i d l y i n t h i s l o t , the dead eggs appeared t o have s u f f e r e d the same f a t e over a l o n g e r p e r i o d o f time, a l l appearing swollen and c l e a r when dead r a t h e r than opaque w h i t e which was t y p i c a l o f dead eggs of the parent species. D u r i n g the 1964 season, the d u r a t i o n o f sperm m o t i l i t y was examined f o r b o t h p a r e n t s p e c i e s . became m o t i l e when p l a c e d i n t o water.  Sperm  A t 18°C., m o t i l i t y  Was l o s t a t the same r a t e i n both s p e c i e s .  From 15-20  seconds a f t e r immersion, t h e r e appeared t o be no r e d u c t i o n in motility.  By 30 seconds, 50% o f t h e sperm was no  l o n g e r moving,  and b y 50-60 seconds, t h e r e was no remaining  motility. A gamete l i f e o f 30 seconds would a l l o w v i a b l e sperm t o d r i f t from 18.3 - 54.9 M. i n the water v e l o c i t i e s observed i n Wolfe Greek.  T h i s equals o r exceeds the  dimensions o f a l l the p o o l s and runs i n which both s p e c i e s were observed so t h a t a c c i d e n t a l f e r t i l i z a t i o n by d r i f t i n g  43 sperm i s a d i s t i n c t p o s s i b i l i t y i n producing  hybrids  in  Wolfe Creek.  D. The Acrocheilus  d i p l o i d number most f r e q u e n t l y recorded  i s 46,  In both cases, J.D.  McPhail  Cytology  and  for Ptychocheilus,  52.  (Table  IV).  these were a l s o the h i g h e s t counts observed.  (per.,  52 f o r C h e h a l i s  and  comm.) observed counts o f both 50 Columbia R i v e r P t y c h o c h e i l u s .  a d i p l o i d number equal t o the sum parents,  o r 49.  h y b r i d r e c i p r o c a l was  the  t o have  o f the h a p l o i d numbers o f  In f a c t , counts of 49 and  most commonly observed, and  and  On  b a s i s o f these counts, one would expect F^ h y b r i d s  the two  for  50 were  appeared t o depend upon which  i n v o l v e d , the female  r e c i p r o c a l showing counts of 50 and  Ptychocheilus  the female A c r o c h e i l u s  r e c i p r o c a l g i v i n g counts o f 49.  y  In c o n s t r u c t i n g karyograms o f the b e s t n u c l e i , there was  c l o s e agreement between d i f f e r e n t n u c l e i from  the same s p e c i e s , such t h a t , when chromosomes were ranked i n descending order a t t o t a l l e n g t h , the morphology o f  the  l  chromosome a t any g i v e n p o s i t i o n i n the rank was one  like  the  from the same p o s i t i o n i n o t h e r n u c l e i o f the same  species,  A t o t a l of s i x Ptychocheilus  and  four  A c r o c h e i l u s n u c l e i were s u f f i c i e n t l y f r e e o f chromosome overlaps  to p e r m i t c o n s t r u c t i o n o f karyograms  ( F i g s . 11,  44  F i g . 11.  C o l c h i c i n e t r e a t e d m i t o t i c metaphase from cultured t e s t i s of Acrocheilus alutaceus. Probable p a i r i n g o f chromosomes i s shown below photograph o f n u c l e u s . 2N = 46.  J \ ( ft 1  2  3  11 11 4  5  II If 6  7  8  ll  II  3  (X-Y?)  It  II II 1 < H  ii  ti  II  II  9  17  10  18  11  19  12  13  ( I IS 20  21  tl  14  22  15  16  T a b l e IV  Frequency o f d i p l o i d chromosome numbers f o r w i l d p a r e n t a l s p e c i e s and reared F, Hybrids  Observed Chromosome Numbers Species  41  42  43  44  45  46  47  48  49  50  6  2  3  8  2  3  51  52  1  10  Acrocheilus alutaceus  Reared Hybrids  ''Female Acrocheilus reciprocal Female Ptychocheilus reciprocal Ptychocheilus oregonensis  45.  46 12 and 13). I f the observed  number o f chromosomes was g r o s s l y  above o r below the a c t u a l number, one would expect cases o f t r i p l e t s o r unpaired  to find  chromosomes, b u t none  occurred. A l l the m a t e r i a l from the p a r e n t s p e c i e s was obtained  from males.  The asymmetrical  p a i r o f chromosomes  (See F i g s . 11 and 12) p o s s i b l y r e p r e s e n t sex chromosomes. Again, McPhail has observed  the same asymmetrical  Chehalis River Ptychocheilus.  There i s no  however, t h a t t h i s p a i r i s heterochromatic, t h e i r meiotic behavior  pair in  evidence, and d a t a on  are l a c k i n g .  Among the l a r g e r chromosomes, i n which the morphology i s d i s t i n c t i v e ,  c l o s e agreement can be seen  between A c r o c h e i l u s and P t y c h o c h e i l u s .  One n o t a b l e  dif-  f e r e n c e i s i n the s m a l l e r o f the two p o s s i b l e sex chromosomes, which i s a v e r y s m a l l m e t a c e n t r i c i n P t y c h o c h e i l u s and a l a r g e r submetacentric  i n Acrocheilus.  Arm numbers c a l c u l a t e d from the karyograms are given i n Table V.  From t h i s i t cannot be shown t h a t any o f the  A c r o c h e i l u s chromosomes r e p r e s e n t c e n t r i c f u s i o n s o f two P t y c h o c h e i l u s chromosomes.  A l s o , t h e r e i s no h i g h e r  p r o p o r t i o n o f n e a r l y symmetrical than  chromosomes i n A c r o c h e i l u s  i n P t y c h o c h e i l u s , which c o u l d i n d i c a t e t r a n s l o c a t i o n s .  I t can o n l y be concluded chromatin.  t h a t P t y c h o c h e i l u s has more  47  F i g . 12,  C o l c h i c i n e t r e a t e d m i t o t i c metaphase from P t y c h o c h e i l u s oregonensis c u l t u r e d t e s t i s . Probable p a i r i n g o f chromosomes i s shown below photograph o f n u c l e u s . 2N = 52.  # H tl tt u 1  2  3  4  5  If  U  ft  It  ii  9  10  11  12  13  ti  M  17  18  at  tt  A^  it  2 5  26  19  (X-Y?)  2 0  »  21  11 6  ui t i 7  *« i t  8  n  14  15  16  it  ti  ii  22  23  24  48  P i g . 13.  C o l c h i c i n e t r e a t e d m i t o t i c metaphases from b r a n c h i a l epithelium of reared F i hybrids. AboveJ female A c r o c h e i l u s r e c i p r o c a l ; 2N = 50. Belows female P t y c h o c h e i l u s r e c i p r o c a l : 2N = 49.  49  Table V  Chromosome arm numbers observed f o r w i l d p a r e n t a l s p e c i e s from Wolfe Lake and the Chehalis River  Species  Observed Arm Number  Acrocheilus alutaceus (Wolfe Lake)  88  Ptychocheilus oregonensis (Wolfe Lake)  98  P t y cho ch e i l u s oregonensis (Chehalis River, McPhail, p e r . comm.)  96  Number o f N u c l e i 4  50  In the absence o f d a t a on m e i o t i c b e h a v i o r o f t h e h y b r i d chromosome the amount o f homology between t h e two s p e c i e s can o n l y be i n f e r r e d .  Because o f the g e n e r a l l y  c l o s e resemblance i n chromosome morphology, i t i s p o s s i b l e t h a t t h e r e i s a h i g h degree o f homology, w i t h the  three  e x t r a P t y c h o c h e i l u s chromosomes a c t i n g as u n i v a l e n t s . F u r t h e r e v i d e n c e f o r a h i g h degree o f homology i s  the  p r e s e n c e o f p r o b a b l e b a c k c r o s s e s i n the p o p u l a t i o n .  If  t h e r e were a l a r g e number o f non homologous chromosomes, t h e n p a i r i n g o f the chromosomes d u r i n g t h e prophase I o f m e i o s i s i n F-^ h y b r i d s would be c o m p l e t e l y b l o c k e d , h y b r i d s would be c o m p l e t e l y s t e r i l e .  and the  S i n c e some F^ h y b r i d s  were o b s e r v e d t o have a t l e a s t p a r t i a l f e r t i l i t y ,  and s i n c e  presumed b a c k c r o s s e s were c o l l e c t e d , a h i g h degree o f chromosomal homology must be assumed.  E . Spawning H a b i t a t and B e h a v i o r A l l the d a t a on n a t u r a l spawning areas o r i g i n a t e from M i s s e z u l a and Wolfe L a k e s .  The l o c a l i t i e s where  fish  i n spawning c o n d i t i o n were o b s e r v e d o r t a k e n are shown i n F i g u r e s 2 and 3 . A c r o c h e i l u s w i t h f r e e gametes were f i r s t t a k e n i n g i l l n e t samples on June 9 d u r i n g t h e 1965 s e a s o n . the 1964 season,  During  t h e y were f i r s t t a k e n on June 12, b u t may  have been t h e r e e a r l i e r .  S u r f a c e water temperature  in a l l  51 r e g i o n s o f the l a k e was 1 8 ° C . when spawning f i s h o f b o t h s p e c i e s were f i r s t o b s e r v e d . D u r i n g c l e a r weather,  mature A c r o c h e i l u s  appeared  t o move i n t o the o u t l e t r e g i o n each n i g h t , and remain t h e r e , s h e l t e r i n g under b r u s h and l o g s d u r i n g the d a y .  Cloudy  weather appeared t o s t o p the movement i n t o the o u t l e t and cause those f i s h w h i c h were t h e r e t o move back i n t o the l a k e . O b s e r v a t i o n s b y d i v i n g d u r i n g the 1964 season showed t h a t a s c h o o l o f A c r o c h e i l u s e s t i m a t e d t o c o n t a i n from 20 t o 50 f i s h was p r e s e n t c l e a r days i n s u c c e s s i o n .  i n the o u t l e t on s e v e r a l  A few P t y c h o c h e i l u s t e n t a t i v e l y  i d e n t i f i e d as males because o f t h e i r s m a l l s i z e and the p r e s e n c e o f w e l l - d e v e l o p e d t u b u r c l e s on the head and pectoral fins,  were i n c l u d e d : i n t h e s c h o o l .  The b e s t  v i s u a l e s t i m a t e o f P t y c h o c h e i l u s abundance was a count o f s i x i n d i v i d u a l s i n a s c h o o l e s t i m a t e d t o c o n t a i n about 50 A c r o c h e i l u s and one h y b r i d . Male P t y c h o c h e i l u s were a l s o t a k e n i n t h e  gillnet  samples from t h e o u t l e t r e g i o n a t t h e r a t e o f one t o about ten A c r o c h e i l u s .  D u r i n g b o t h seasons,  only three sexually  mature female P t y c h o c h e i l u s were t a k e n from the  outlet.  P t y c h o c h e i l u s i n M i s s e z u l a Lake were o b s e r v e d t o spawn i n an a r e a along; the west shore o f the l a k e , 0.8 Km. n o r t h o f t h e o u t l e t r e g i o n ( F i g . 2 ) .  about  Sexually  mature male P t y c h o c h e i l u s were a l s o found i n the o u t l e t o f M i s s e z u l a Lake a t t h e same t i m e t h a t A c r o c h e i l u s began t o  52 appear, a l t h o u g h spawning i n P t y c h o c h e i l u s was n o t o b s e r v e d u n t i l June 21 d u r i n g the 1965 s e a s o n . p r o b a b l y due t o d i f f i c u l t y localities,  T h i s , however, was  i n f i n d i n g P t y c h o c h e i l u s spawning  s i n c e a mature female was t a k e n i n the o u t l e t on  June 12 d u r i n g t h e 1964 season and on June 15 d u r i n g the 1965 s e a s o n . Spawning was o b s e r v e d on f o u r days d u r i n g the 1965 season,  each time a t dusk,  dense mass o f f i s h , size,  from 7 . 3 0 p . m . t o 10 p . m .  apparently males,  A  j u d g i n g from t h e i r  o c c u p i e d the areai i m m e d i a t e l y o f f s h o r e .  Some o f  f i s h c l o s e s t t o s h o r e were m o s t l y o u t o f t h e w a t e r . d i m e n s i o n s o f the a g g r e g a t i o n were about 3.1 M . from  The inshore  t o o f f s h o r e e x t r e m i t y and about 4 . 6 M . p a r a l l e l t o t h e I n t h e deeper w a t e r , larger fish,  the  shore.  o f f s h o r e from the a g g r e g a t i o n o f males,  a l m o s t c e r t a i n l y f e m a l e s , were swimming around  t h e p e r i p h e r y o f the a g g r e g a t i o n .  Females a p p a r e n t l y  entered, t h e a g g r e g a t i o n n e a r t h e s h o r e ,  and spawning took  p l a c e i n water too shallow to cover the f i s h , b y much s p l a s h i n g b y t h e m a l e s .  accompanied  I n a few c a s e s ,  the  female  was s u f f i c i e n t l y v i s i b l e t o see h e r body v i b r a t i n g , presumably d u r i n g e m i s s i o n o f the eggs.  On one o c c a s i o n the  a g g r e g a t i o n d i s p e r s e d due t o d i s t u r b a n c e b u t r e - f o r m e d i n about 30 m i n u t e s .  At nightfall,  the s c h o o l d i s p e r s e d ,  and  no f u r t h e r a c t i v i t y was h e a r d o r seen when o b s e r v a t i o n s were extended t o one h o u r a f t e r  nightfall.  No spawning a c t i v i t y  was o b s e r v e d d u r i n g t h e e a r l y m o r n i n g , j u s t a f t e r dawn.  52 appear, a l t h o u g h spawning i n P t y c h o c h e i l u s was n o t o b s e r v e d u n t i l June 21 d u r i n g the 1965 s e a s o n . p r o b a b l y due t o d i f f i c u l t y localities,  T h i s , however, was  i n f i n d i n g P t y c h o c h e i l u s spawning  s i n c e a mature female was t a k e n i n the o u t l e t on  June 12 d u r i n g the 1964 season and on June 15 d u r i n g the 1965 s e a s o n . Spawning was o b s e r v e d on f o u r days d u r i n g the 1965 season,  each time a t dusk,  dense mass o f f i s h , size,  from 7 . 3 0 p . m . t o 10 p . m .  a p p a r e n t l y males,  A  j u d g i n g from t h e i r  o c c u p i e d the a r e a i m m e d i a t e l y o f f s h o r e .  Some o f  f i s h c l o s e s t t o shore were m o s t l y o u t o f the w a t e r . d i m e n s i o n s o f the a g g r e g a t i o n were about 3.1 M . from  The inshore  t o o f f s h o r e e x t r e m i t y and about 4 . 6 M . p a r a l l e l t o t h e I n t h e deeper w a t e r , larger fish,  the  shore.  o f f s h o r e from the a g g r e g a t i o n o f males,  a l m o s t c e r t a i n l y females, were swimming around  t h e p e r i p h e r y o f the a g g r e g a t i o n .  Females a p p a r e n t l y  e n t e r e d t h e a g g r e g a t i o n near the s h o r e ,  and spawning took  p l a c e i n water too shallow to cover the f i s h , b y much s p l a s h i n g b y t h e m a l e s .  accompanied  I n a few c a s e s ,  the  female  was s u f f i c i e n t l y v i s i b l e t o see h e r body v i b r a t i n g , presumably d u r i n g e m i s s i o n o f the eggs.  On one o c c a s i o n t h e  a g g r e g a t i o n d i s p e r s e d due t o d i s t u r b a n c e b u t r e - f o r m e d i n about 30 m i n u t e s . no f u r t h e r  At n i g h t f a l l ,  the s c h o o l d i s p e r s e d ,  and  a c t i v i t y was h e a r d o r seen when o b s e r v a t i o n s were  extended t o one hour a f t e r  nightfall.  No spawning a c t i v i t y  was o b s e r v e d d u r i n g t h e e a r l y m o r n i n g , j u s t a f t e r dawn.  53 E x a m i n a t i o n o f the, spawning l o c a l i t y r e v e a l e d l a r g e numbers o f eggs, a l l v e r y c l o s e t o s h o r e , 15 cm. o f w a t e r .  in less  The a r e a o f egg d e p o s i t i o n extended  about 3.6 M. a l o n g the  than for  shore.  G i l l n e t c o l l e c t i o n s from t h i s l o c a l i t y y i e l d e d l a r g e numbers o f r i p e male P t y c h o c h e i l u s and s m a l l e r numbers o f females  i n n e a r l y mature, r i p e ,  condition.  or r e c e n t l y  spent  O n l y one A c r o c h e i l u s , a s p e n t male,  was  c o l l e c t e d i n t h i s a r e a near the end o f the spawning season (July 12). P t y c h o c h e i l u s p r o b a b l y spawns i n o t h e r areas o f the l a k e , b u t t h e s e were n o t l o c a t e d d u r i n g the c o u r s e o f the s t u d y .  I t cannot be shown c o n c l u s i v e l y t h a t  A c r o c h e i l u s spawns o n l y i n the- o u t l e t r e g i o n . common f i s h occurrence  i n the l a k e , however,  I t i s not a  and i t s h i g h r a t e o f  i n the o u t l e t r e g i o n d u r i n g the spawning season  would seem t o be s u f f i c i e n t t o account f o r the spawning o f the e n t i r e l a k e p o p u l a t i o n . I n W o l f e L a k e , b o t h A c r o c h e i l u s and P t y c h o c h e i l u s u t i l i z e d the i n l e t stream, Underwater o b s e r v a t i o n s  W o l f e Greek,  for  spawning.  showed A c r o c h e i l u s and P t y c h o c h e i l u s  t o be abundant i n the deeper p o o l s and runs i n the h a l f o f the s t r e a m ' s l e n g t h .  A large portion of  lower the  r e m a i n i n g d i s t a n c e upstream t o I z z i t ' s Lake c o n s i s t e d o f straight  runs w i t h l i t t l e c o v e r and a l m o s t no f i s h .  For  about 91.4 meters below I z z i t ' s L a k e , p o o l s were p r e s e n t ,  54 occupied  again by A c r o c h e i l u s and P t y c h o c h e i l u s .  These  fish  may, however, have o r i g i n a t e d from I z z i t ' s Lake r a t h e r than Wolfe Lake. U n l i k e M i s s e z u l a Lake, there was no s p a t i a l s e g r e g a t i o n o f A c r o c h e i l u s and P t y c h o c h e i l u s i n Wolfe Creek. Both were abundant wherever c o n d i t i o n s were s u i t a b l e . the A c r o c h e i l u s appeared t o form groups which moved independent o f the P t y c h o c h e i l u s .  Non-sexual  Also,  together,  interactions,  p r i m a r i l y nudging, chasing, and grouping together,  appeared  to be c o n f i n e d t o w i t h i n each s p e c i e s and no i n t e r s p e c i f i c a c t s were observed.  This i s i n contrast to observations  i n the o u t l e t o f M i s s e z u l a Lake, where the P t y c h o c h e i l u s males a c t i v e l y o r i e n t e d themselves with,  and moved about  with Acrocheilus. Spawning was n o t observed i n Wolfe Creek, b u t eggs c o l l e c t e d from two o f the runs where b o t h p a r e n t were p r e s e n t were reared i n the l a b o r a t o r y .  The f r y  produced were m o s t l y P t y c h o c h e i l u s , b u t a number o f A c r o c h e i l u s were a l s o p r e s e n t .  species  55  DISCUSSION A. O r i g i n o f the W i l d  Intermediates  Four l i n e s o f evidence  a r e a v a i l a b l e which  i n d i c a t e t h a t the w i l d i n t e r m e d i a t e s a r e the r e s u l t o f h y b r i d i z a t i o n between P t y c h o c h e i l u s and A c r o c h e i l u s , and t h a t most o f them a r e F^ h y b r i d s .  First,  the comparative  morphology o f the w i l d i n t e r m e d i a t e s and the p a r e n t a l s p e c i e s shows t h a t the i n t e r m e d i a t e s ,  f o r the most p a r t ,  l i e between the v a l u e s f o r the p a r e n t s examined. squawfish  i n the c h a r a c t e r s  There would appear t o be some dominance o f f e a t u r e s , however, s i n c e the i n t e r m e d i a t e s  lack  the k e r a t i n i z e d margin o f the lower jaw, t y p i c a l l y show two  t e e t h on the outer, pharyngeal  t o o t h row, and l a c k the  c o i j i n g t y p i c a l o f t h e chiselmouth g u t . Second, w i t h the e x c e p t i o n o f the disagreement i n o u t e r row pharyngeal  t o o t h counts i n the female A c r o c h e i l u s  r e c i p r o c a l s , the r e a r e d F^ h y b r i d s a r e s i m i l a r i n a l l r e s p e c t s t o the w i l d  intermediates.  T h i r d , the o f f s p r i n g o f the b a c k c r o s s  experiment,  i n which the a b e r r a n t male from M i s s e z u l a Lake was c r o s s e d t o a female A c r o c h e i l u s , show a pharyngeal  tooth count  s e g r e g a t i o n t h a t can o n l y be e x p l a i n e d b y assuming t h a t the male had chiselmouth t y p i c a l intermediates  genes.  T h i s male shared  with  the dark peritoneum and the l o n g  a n t e r i o r f o l d o f the i n t e s t i n e .  It differed  i n having a  56 straight, count.  r a t h e r than d e c u r v e d mouth and i n a l o w e r s c a l e I t d i f f e r e d from presumed M y l o c h e i l u s x P t y c h o c h e i l u s  h y b r i d s from M i s s e z u l a Lake i n t h a t the l a t t e r have a s i l v e r y peritoneum,  a s h o r t a n t e r i o r f o l d o f the i n t e s t i n e  l i k e Mylocheilus,  and,  the v e n t r a l two p h a r y n g e a l t e e t h o f  the  i n n e r row a r e s h o r t and b l u n t r a t h e r than p o i n t e d as i n A c r o c h e i l u s , P t y c h o c h e i l u s , and i n t h e M i s s e z u l a Lake aberrant male.  T h i s male a l s o d i f f e r e d from the  reared  h y b r i d s between P t y c h o c h e i l u s and R i c h a r d s o n i u s i n t h a t latter  show a s i l v e r y p e r i t o n e u m w i t h d i f f u s e  a h i g h a n a l r a y count  the  melanophores,  (11, as opposed t o 7, 8 o r 9 f o r  P t y c h o c h e i l u s and A c r o c h e i l u s i n M i s s e z u l a Lake) and i n t h e more o b l i q u e mouth i n P t y c h o c h e i l u s x R i c h a r d s o n i u s h y b r i d s . T h i s l e a v e s open o n l y two p o s s i b i l i t i e s ;  e i t h e r the  aberrant  male was an u n u s u a l P t y c h o c h e i l u s , o r was o f h y b r i d o r i g i n . I t has been shown t h a t r e a r e d F^ h y b r i d s and w i l d  inter-  mediates always have a t l e a s t one o u t e r row p h a r y n g e a l tooth.  I f t h e male i n q u e s t i o n were an a b e r r a n t  Ptychocheilus,  t h e n the same r e s u l t would be e x p e c t e d upon  c r o s s i n g him t o a female A c r o c h e i l u s .  The f a c t t h a t 62  p e r c e n t o f the o f f s p r i n g showed a 0-0 count e x c l u d e s  the  p o s s i b i l i t y t h a t he was an a b e r r a n t P t y c h o c h e i l u s , l e a v i n g o n l y h y b r i d o r i g i n as an e x p l a n a t i o n . Fourth, the w i l d intermediates sterile.  are p a r t i a l l y  Of t h o s e w h i c h d e v e l o p sex p r o d u c t s ,  the  females  appear to produce i n v i a b l e eggs and about 40 p e r c e n t o f  57  p a r e n t a l eggs f e r t i l i z e d w i t h sperm from i n t e r m e d i a t e males show abnormal cleavage  and d i e .  These o b s e r v a t i o n s  the p o s s i b i l i t y t h a t the w i l d i n t e r m e d i a t e s might be r a r e and h i t h e r t o unrecognized  s p e c i e s which  exclude a  reproduces  itself. The p r o b a b i l i t y t h a t most o f the w i l d h y b r i d s  are  P-j^s . i s i n d i c a t e d by t h e i r agreement i n morphology w i t h r e a r e d F-^s, and  the observed  wild hybrids.  Because of t h e i r low abundance and  degree of s t e r i l i t y , i s extremely  low  h i g h degree o f s t e r i l i t y i n  the chances o f h y b r i d t o h y b r i d matings  (Hubbs, 1 9 5 5 ) i f not absent  view o f the apparent  altogether i n  t o t a l i n v l a b i l i t y o f h y b r i d eggs.  I f o t h e r than F ^ h y b r i d s are to occur,  they must  c e r t a i n l y be the r e s u l t o f b a c k c r o s s i n g t o one parent  species.  In f a c t ,  two  or the  other of  t h i s type of o r i g i n .  from M i s s e z u l a Lake might be  P t y c h o c h e i l u s backcrosses,  almost  the f o u r w i l d i n t e r m e d i a t e s  a b e r r a n t morphology seem t o suggest The  high  i n t e r p r e t e d as F ^ x  w h i l e the two Wolfe Lake  examples might be F]_ x A c r o c h e i l u s b a c k c r o s s e s .  I t seems  c e r t a i n t h a t the M i s s e z u l a Lake i n d i v i d u a l s are o f h y b r i d origin,  from the argument o u t l i n e d above.  between these  The d i f f e r e n c e s  i n d i v i d u a l s and t y p i c a l h y b r i d s are i n the  more P t y c h o c h e i l u s - 1 i k e v a l u e s f o r some c h a r a c t e r s . o f t h i s , FJL t o P t y c h o c h e i l u s b a c k c r o s s e s the most l o g i c a l e x p l a n a t i o n of t h e i r  Because  would seem t o be  origin.  The Wolfe Lake a b e r r a n t s show a morphology  58 i n t e r m e d i a t e between an suggest b a c k c r o s s i n g  and A c r o c h e i l u s , w h i c h would  w i t h A c r o c h e i l u s as t h e i r o r i g i n .  e v i d e n c e i n t h i s case i s not as d i r e c t , however. i s known t o h y b r i d i z e o n l y w i t h P t y c h o c h e i l u s ,  The  Acrocheilus  but  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  a r e a l s o i n the same a r e a  d u r i n g the spawning p e r i o d .  An A c r o c h e i l u s male x  Richardsonius  female c r o s s was made a t Wolfe l a k e u s i n g 10  Richardsonius  eggs.  One i n d i v i d u a l s u r v i v e d t o a s i z e  w h i c h s c a l e s were formed,  and i t s morphology  from the Wolfe Lake a b e r r a n t s .  is  at  different  The p o s s i b l e morphology  Acrocheilus x Mylocheilus i s a speculative matter.  In  of the  two A c r o c h e i l u s h y b r i d c r o s s e s w h i c h were made a r t i f i c i a l l y however, the k e r a t i n i z e d surpressed,  as was the  edge on the l o w e r jaw was  gut c o i l i n g .  totally  I f an A c r o c h e i l u s x  M y l o c h e i l u s c r o s s c o u l d be expected t o behave s i m i l a r l y , t h e n the Wolfe Lake a b e r r a n t s would d i f f e r a keratinized  s i n c e t h e y have  margin on the lower jaw and an i n f l e c t e d  gut.  B . Causes o f H y b r i d i z a t i o n In a l l known h y b r i d l o c a l i t i e s L a k e , the h y b r i d s  are r e l a t i v e l y  rare.  except M i s s e z u l a Wolfe Lake, w i t h a  1.3 per c e n t r e l a t i v e abundance o f h y b r i d s figure  i s the  r e c o r d e d e x c e p t f o r M i s s e z u l a L a k e , where  abundance o f h y b r i d s  i s 13.2 p e r c e n t r e l a t i v e t o  The l a r g e gap between M i s s e z u l a Lake and a l l the  highest the Acrocheilus. other  59 h y b r i d l o c a l i t i e s i n r e l a t i v e abundance o f h y b r i d s s u g g e s t s t h a t two d i f f e r e n t mechanisms may be o p e r a t i n g t o cause hybr i d i z a t i o n . In M i s s e z u l a L a k e , b o t h s p e c i e s spawn i n the A c r o c h e i l u s uses the o u t l e t narrows above the dam,  lake.  apparently  t o the e x c l u s i o n o f a l l o t h e r a r e a s , and P t y c h o c h e i l u s spawns i n the main body o f the l a k e .  Some s e x u a l l y mature  P t y c h o c h e i l u s a r e found i n t h e o u t l e t r e g i o n , away from t h e i r observed spawning a r e a .  Because o f t h i s ,  the s c h o o l  i n t h e o u t l e t i s m i x e d , c o n t a i n i n g m o s t l y s e x u a l l y mature A c r o c h e i l u s and a few, m o s t l y mature male P t y c h o c h e i l u s . P t y c h o c h e i l u s was observed t o be a group  spawner,  w i t h a number o f males spawning w i t h one o r a few females a t the same t i m e .  S c h u l t z (1935) observed a s i m i l a r  spawning h a b i t i n M y l o c h e i i u s , and i n Wolfe C r e e k , R i c h a r d s o n i u s was o b s e r v e d t o spawn i n ' l a r g e groups w i t h no a p p a r e n t p a i r f o r m a t i o n . spawning, b u t the p a t t e r n  A c r o c h e i l u s was n o t  i s presumably s i m i l a r t o  o b s e r v e d f o r the o t h e r t h r e e r e l a t e d  seen that  genera."  I f group spawning i s assumed f o r A c r o c h e i l u s , t h e n h y b r i d i z a t i o n may a r i s e i n one o r b o t h o f the ing ways.  F i r s t , s i n c e t h e r e a r e few male P t y c h o c h e i l u s  s c h o o l i n g w i t h the A c r o c h e i l u s i n the o u t l e t , participate female.  follow-  these might  i n f e r t i l i z a t i o n o f the eggs o f an A c r o c h e i l u s  Second, s i n c e female P t y c h o c h e i l u s w i t h f r e e  o c c u r o c c a s i o n a l l y i n the o u t l e t ,  eggs  t h e y may a l s o spawn t h e r e .  60 I n the case o f the M i s s e z u l a Lake o u t l e t ,  the  o n l y male P t y c h o c h e i l u s a v a i l a b l e are s c h o o l i n g w i t h Acrocheilus,  the  and i t would seem, on the b a s i s o f r e l a t i v e  numbers o f the two s p e c i e s p r e s e n t  i n the o u t l e t ,  that a  spawning female P t y c h o c h e i l u s would more l i k e l y h y b r i d i z e than spawn w i t h a male o f h e r own s p e c i e s . d i f f e r e n c e i n p h a r y n g e a l t o o t h counts between  If  the  reared  female A c r o c h e i l u s and female P t y c h o c h e i l u s r e c i p r o c a l c r o s s e s i s c o n s i s t e n t i t i n d i c a t e s t h a t the b u l k o f w i l d h y b r i d s a r e from P t y c h o c h e i 1us eggs.  Possibly,  hybridiz-;  a t i o n v i a female P t y c h o c h e i l u s i s the b e s t e x p l a n a t i o n f o r t h e h i g h r e l a t i v e abundance o f h y b r i d s i n M i s s e z u l a L a k e . I f h y b r i d i z a t i o n was o c c u r i n g o n l y through mixed males f e r t i l i z i n g female A c r o c h e i l u s ,  the n u m e r i c a l l y l e s s  abundant P t y c h o c h e i 1 u s males c o u l d f e r t i l i z e o n l y a s m a l l p r o p o r t i o n o f the eggs o f any female, and a d u l t h y b r i d s would be l e s s abundant than o b s e r v e d .  On the o t h e r hand,  a female P t y c h o c h e i l u s i n t h e same s c h o o l o f f i s h would l o s e most o f h e r eggs t o - h y b r i d i z a t i o n because o f greater  the  abundance o f male A c r o c h e i l u s . Hubbs, W a l k e r and Johnson (1943), and Hubbs,  Hubbs and Johnson (1943) r e p o r t cases where i n t e r s p e c i f i c h y b r i d i z a t i o n o f a t h e r i n i d s and c a t o s t o m i d s r e s p e c t i v e l y a r e p r o b a b l y due t o a d i s p r o p o r t i o n a t e r e l a t i v e o f the p a r e n t a l s p e c i e s .  abundance  Mayr (1963) a l s o c i t e s cases  i n b i r d s where zones o f c o n t a c t e s t a b l i s h e d on t h e p e r i p h e r y  61 of  the range o f one p a r e n t a l form can l e a d t o h y b r i d i z a t i o n .  This a l s o applies to rare strays  i n n o r m a l l y segregated  r e p r o d u c t i v e groups w i t h i n a s m a l l a r e a .  The s t r a y s , b e i n g  w i t h i n the m a t i n g a r e a o f the o t h e r s p e c i e s , w i l l be u n a b l e t o f i n d c o n s p e c i f i c mates and w i l l h y b r i d i z e (Mayr, 1 9 6 3 ) . In Wolfe C r e e k , the c i r c u m s t a n c e s a r e different.  quite  Here t h e r e i s no o b s e r v a b l e s e g r e g a t i o n o f  s e x u a l l y mature A c r o c h e i l u s and P t y c h o c h e i l u s .  Both  species  a r e c o n c e n t r a t e d i n the deeper p o o l s and r u n s , u s i n g whate v e r top c o v e r i s a v a i l a b l e .  Spawning was n o t o b s e r v e d  i n Wolfe Creek, b u t b o t h s p e c i e s must spawn i n t h e same areas where the a g g r e g a t i o n s were seen,  s i n c e eggs t a k e n  from t h o s e a r e a s and r e a r e d p r o v e d t o b e l o n g t o b o t h s p e c i e s . S i n c e the r e l a t i v e abundance o f a d u l t h y b r i d s i s much l o w e r i n Wolfe Lake t h a n i n M i s s e z u l a L a k e ,  species  r e c o g n i t i o n must t a k e p l a c e , s i n c e t h e r e i s no s p a t i a l segregation.  The r e l a t i v e abundance o f A c r o c h e i l u s and  P t y c h o c h e i l u s as e s t i m a t e d from v i s u a l counts o f s e x u a l l y mature f i s h  i n Wolfe C r e e k , i s n e a r l y e q u a l .  In  i n the o u t l e t o f M i s s e z u l a Lake, P t y c h o c h e i l u s i s r e l a t i v e to Acrocheilus, The  contrast, rare  and s c h o o l s w i t h A c r o c h e i l u s .  o n l y o b s e r v a t i o n s made i n Wolfe Creek which  a r e a p p l i c a b l e t o the p r o b l e m o f mate s e l e c t i o n i n the two species are that,  i n the aggregations,  to c l u s t e r together,  t h e A c r o c h e i l u s seem  r a t h e r than b e i n g s c a t t e r e d  among the P t y c h o c h e i l u s .  Also,  uniformly  the o n l y i n t e r a c t i o n s  62 which were observed were i n t r a s p e c i f i c ,  suggesting that  species recognition is operating, at least in non-sexual interactions. If,  in fact,  species recognition and segretation  are operative when, r e l a t i v e abundance is more nearly equal, then chance encounters of d r i f t i n g gametes may account for the l e v e l of hybridization observed.  From experiments on  sperm of Missezula Lake Acrocheilus and Ptychocheilus  it  would appear that the sperm can be in contact with water for 15 to 20 seconds before m o t i l i t y begins to decline. At the water v e l o c i t i e s measured in Wolfe Creek in areas where sexually mature f i s h were observed, sperm could d r i f t from 4.6 to 36.3 meters, depending on the water v e l o c i t y before their m o t i l i t y began to decline.  Since  the two species were observed to be closer together than this,  and since eggs of both were taken from the same area,  chance f e r t i l i z a t i o n probably does occur.  This could be  a general phenomenon, since the spawning habitat used by the two species is similar i n most  respects.  Hubbs (1957, 1960 and 1961) has studied duration of sperm function and gamate compatibility as related to i n t e r s p e c i f i c hybrid formation.  The delay times he  observed before f e r t i l i z a t i o n percentages began to decline are comparable to the delay times observed for Acrocheilus and Ptychocheilus before m o t i l i t y declined sharply.  63  C. Consequences o f H y b r i d i z a t i o n Swamping o f e i t h e r p a r e n t a l form has n o t o c c u r r e d , even where the r a t e o f h y b r i d i z a t i o n i s as h i g h as i n Missezula Lake.  I f i n t r o g r e s s i o n has t a k e n p l a c e ,  n o t r e v e a l e d i n the morphology o f e i t h e r p a r e n t  it  is  species.  The w i l d p o p u l a t i o n s appear t o c o n s i s t o f d i s t i n c t and unaltered parent species, with intermediates, mostly F i h y b r i d s appearing i n s c a t t e r e d l o c a l i t i e s throughout s y m p a t r i c range o f the p a r e n t  the  species.  S i n c e the F ^ h y b r i d s a r e p a r t i a l l y f e r t i l e , and r a r e b a c k c r o s s e s are known, b a r r i e r s o t h e r than  sterility  must p r e v e n t gene f l o w between t h e p a r e n t s p e c i e s .  The  low abundance and p a r t i a l s t e r i l i t y o f the h y b r i d s , combined w i t h t h e s t r o n g m o r p h o l o g i c a l and e c o l o g i c a l d i v e r g e n c e o f t h e p a r e n t s p e c i e s , c o u l d o p e r a t e t o l i m i t the r a t e which b a c k c r o s s i n g t a k e s p l a c e , and a l s o , t o  at  restrict  s e v e r e l y the s u r v i v a l o f b a c k c r o s s o f f s p r i n g because o f t h e i r u n b a l a n c e d and n o n - a d a p t i v e  genotypes.  Swamping o r i n t r o g r e s s i o n can t a k e p l a c e o n l y i f the h y b r i d s themselves are a t a s e l e c t i v e advantage,  or i f  c e r t a i n gene complexes o f one p a r e n t a r e a t a s e l e c t i v e advantage t o t h e i r a l t e r n a t i v e s (Anderson,  1 9 5 3 , Mayr,  i n the o t h e r  1 9 6 3 , Bigelow,  1965).  parent In order for  F-L h y b r i d s t o s u r v i v e a t a l l , t h e r e must be a s u i t a b l e environment open t o them.  F o r h y b r i d g e n e r a t i o n s beyond  64 t h e F-^, o r f o r b a c k c r o s s e s ,  r e c o m b i n a t i o n w i l l cause much  greater v a r i a b i l i t y than i n F i h y b r i d s , w i t h the  result  t h a t many recombinants w i l l be i n v i a b l e o r p o o r l y a d a p t e d . I n organisms which a r e w i d e l y d i v e r g e n t ,  the number o f  p o s s i b l e a d a p t i v e recombinants would be o n l y a s m a l l f r a c t i o n o f t h e t o t a l number o f p o s s i b l e r e c o m b i n a n t s . T h i s would s e v e r e l y r e s t r i c t the s u r v i v a l . o f b a c k c r o s s offspring,  o r o f h y b r i d g e n e r a t i o n s beyond t h e F ^ , though  s u r v i v a l o f F^s may be good. S i n c e t h e F-^ h y b r i d s are n u m e r i c a l l y r a r e , w i l l p r o b a b l y b a c k c r o s s t o one o f the p a r e n t r a t h e r than m a t i n g w i t h one another  (Hubbs,  species,  Also,  It  1 9 5 5 ) .  been shown t h a t P t y c h o c h e i l u s i s a mass-spawner, Acrocheilus probably i s .  they  has  and t h a t  the e v i d e n c e i n d i c a t e s i n  female F^ h y b r i d s , any eggs w h i c h may be produced a r e inviable.  The o n l y c h a n n e l open f o r gene f l o w ,  then,  would be the male F ^ s , o f w h i c h o n l y 3 out o f 20 examined had f r e e gametes.  I n o r d e r f o r a male F^ t o f e r t i l i z e  any  p a r e n t a l eggs, he would have t o compete w i t h a r e l a t i v e l y l a r g e number o f A c r o c h e i l u s o r P t y c h o c h e i l u s males which were spawning w i t h t h e same female s i m u l t a n e o u s l y . best,  then,  At  a male h y b r i d c o u l d f e r t i l i z e o n l y a s m a l l  p o r t i o n o f t h e eggs o f a g i v e n f e m a l e .  From the  evidence  g i v e n , about 40 p e r c e n t o f t h e s e eggs would d i e b e f o r e c l e a v a g e was c o m p l e t e .  I f a l l the remainder h a t c h e d ,  t h e r e would s t i l l be heavy m o r t a l i t y i n the o f f s p r i n g f o r  65 the reasons o u t l i n e d above. Only one b a c k c r o s s , t h e second M i s s e z u l a Lake male, had  f r e e gametes.  The f i r s t M i s s e z u l a Lake b a c k c r o s s was  c o l l e c t e d on 26th J u l y , o r about two weeks a f t e r spawning i n b o t h p a r e n t s p e c i e s was f i n i s h e d i n the two seasons o f o b s e r v a t i o n , and appeared  t o be unspawned.  N e i t h e r o f the  Wolfe Lake backcrosses had f r e e eggs, although the o v a r i e s of both appeared  g r o s s l y normal and w e l l  developed.  The second M i s s e z u l a Lake b a c k c r o s s male produced sperm w i t h h i g h f e r t i l i t y ,  b u t t h i s does n o t mean t h a t a l l  b a c k c r o s s e s would n e c e s s a r i l y be f e r t i l e . of  While t h e l a c k  f r e e eggs i n Wolfe Lake b a c k c r o s s females might mean  o n l y t h a t they had n o t y e t completed  maturation, t h e  unspawned c o n d i t i o n o f t h e f i r s t M i s s e z u l a Lake male b a c k c r o s s i s p o s i t i v e evidence t h a t he was somehow b a r r e d from r e p r o d u c t i o n .  Post-spawning  s p e c i e s a l l had spent gonads. developed  samples o f b o t h p a r e n t  The presence o f the w e l l -  gonads i n t h i s backcross i s e x c e p t i o n a l . A p p a r e n t l y i n t r o g r e s s i o n o r swamping a r e com-  p l e t e l y b l o c k e d i n both d i r e c t i o n s . m a i n t a i n themselves  The p a r e n t s p e c i e s  as d i s t i n c t and separate p o p u l a t i o n s  w i t h no gene exchange, and the gametes which e n t e r i n t o h y b r i d i z a t i o n are l o s t . None o f the a v a i l a b l e evidence i n d i c a t e s whether the r a t e o f h y b r i d i z a t i o n i s s t a t i c , manner.  o r v a r y i n g i n some  I f the gamete wastage were s i g n i f i c a n t ,  isolating  66  mechanisms might be expected t o have developed which would p r e v e n t h y b r i d i z a t i o n , b u t t h i s does n o t seem t o be the case.  In a l l the areas s t u d i e d , P t y c h o c h e i l u s  population  l e v e l s a r e h i g h enough t h a t wastage due t o h y b r i d i z a t i o n i s probably  not s i g n i f i c a n t .  In the case o f M i s s e z u l a  Lake, A c r o c h e i l u s i s s u f f i c i e n t l y r a r e f o r the observed l e v e l o f h y b r i d i z a t i o n t o a f f e c t i t s abundance. not imply,  T h i s does  however, t h a t the r a r i t y o f A c r o c h e i l u s i s due t o  hybridization.  M i s s e z u l a Lake a f f o r d s o n l y  marginal  h a b i t a t f o r a bottom-feeding c y p r i n i d such as A c r o c h e i l u s because o f the r e s t r i c t e d l i t t o r a l area.  The r e l a t i v e l y  r e c e n t damming o f the l a k e may a l s o have a f f e c t e d a d v e r s e l y the s u r v i v a l o f eggs and young, s i n c e no o t h e r stream i s a v a i l a b l e f o r spawning.  H y b r i d i z a t i o n alone,  then, cannot  account f o r the low abundance o f A c r o c h e i l u s i n Missezula Lake but  f u r t h e r drops i n the p o p u l a t i o n l e v e l ,  i f they do occur,  m a y b e due t o gamete wastage from h y b r i d i z a t i o n . o t h e r areas where h y b r i d s have been c o l l e c t e d ,  In the  the abundance  o f A c r o c h e i l u s i s much g r e a t e r , and the i n c i d e n c e o f h y b r i d i z a t i o n much lower. Since h y b r i d s have been taken i n w i d e l y s c a t t e r e d localities,  and were known b e f o r e the Columbia R i v e r System  had been m o d i f i e d by man, i t can be concluded  that  h y b r i d i z a t i o n a t a low l e v e l has taken p l a c e between A c r o c h e i l u s and P t y c h o c h e i l u s into  contact.  s i n c e the two s p e c i e s came  67  D. R e l a t i o n s h i p o f P t y c h o c h e i l u s and  Acrocheilus  A c r o c h e i l u s and P t y c h o c h e i l u s are s t r o n g l y d i v e r g e n t i n t h e i r morphology and ecology, b u t much o f the  evidence  c o l l e c t e d d u r i n g the p r e s e n t study i n d i c a t e s t h a t they rather closely related genetically.  Several  are  morphological  c h a r a c t e r s , e s p e c i a l l y the k e r a t i n i z e d margin of. the lower jaw,  the gut c o i l i n g ,  and the pharyngeal  tooth counts,  p a r t i a l o r complete dominance e f f e c t s i n backcrosses.  hybrids  A l s o , the myogen electropherograms  show any c o n s i s t e n t d i f f e r e n c e s between the two. s i n g l e evidence  and  failed The  to  best  o f g e n e t i c s i m i l a r i t y , however, i s the  i n d i c a t i o n o f p a r t i a l h y b r i d f e r t i l i t y and wild  show  the presence of  backcrosses. It  artificially  i s p o s s i b l e to produce v i a b l e F^ h y b r i d s from p a r e n t s w i t h s t r o n g l y d i v e r g e n t genotypes.  Such h y b r i d s can a l s o be a h y b r i d to be  fertile,  found  i n the w i l d .  however, t h e r e must be  In o r d e r f o r sufficient  homology between the p a r e n t a l chromosome complements t o a l l o w proper p a i r i n g of the chromosomes d u r i n g prophase I of  meiosis.  Even r e l a t i v e l y small d i f f e r e n c e s can l e a d to  partial infertility, normally w i l l  s i n c e f a i l u r e o f chromosomes t o p a i r  cause them to behave abnormally  during  the  metaphase and anaphase p o r t i o n s o f the f i r s t m e i o t i c division  ( S i n o t t , Dunn and Dobzhansky, 1958;  Darlington,  1958).  T h i s r e s u l t s i n the secondary gametocytes  68 r e c e i v i n g i m p e r f e c t s e t s o f chromosomes, them i n v i a b l e .  and thus  renders  Because o f the mechanics o f chromosome  p a i r i n g i n v o l v e d i n s u c c e s s f u l m e i o s i s , the f a c t t h a t A c r o c h e i l u s x P t y c h o c h e i l u s h y b r i d s are capable o f o c c a s i o n a l l y p r o d u c i n g v i a b l e gametes i n d i c a t e s a h i g h degree o f chromosomal homology. F u r t h e r evidence o f g e n e t i c s i m i l a r i t y i s p r o v i d e d by the morphology o f the chromosomes. noted  I t has been  e a r l i e r that i n r e a d i l y i d e n t i f i a b l e p a i r s of  chromosomes,  t h e r e i s c l o s e comparison i n the morphology  o f these p a i r s between A c r o c h e i l u s and P t y c h o c h e i l u s . J.D. McPhail  (per. comm.) has found t h a t t h i s  similarity  can a l s o be seen 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 chromosomes . The a v a i l a b l e evidence,  then,  suggests  a close  g e n e t i c s i m i l a r i t y , n o t o n l y between A c r o c h e i l u s and P t y c h o c h e i l u s , b u t a l s o these two and R i c h a r d s o n i u s and Mylocheilus.  A l l permutations  o f h y b r i d s between these  f o u r genera except A c r o c h e i l u s x M y l o c h e i l u s and A c r o c h e i l u s x R i c h a r d s o n i u s a r e known t o occur  naturally.  The q u e s t i o n o f the taxonomic p o s i t i o n o f A c r o c h e i l u s and P t y c h o c h e i l u s i s , however, a s u b j e c t i v e matter.  Obviously,  they meet a l l the c r i t e r i a o f good  s p e c i e s , and t h i s has never been q u e s t i o n e d . should be regarded decision.  Whether  they  as d i s t i n c t genera i s an a r b i t r a r y  I f t o t a l h y b r i d s t e r i l i t y were the c r i t e r i o n ,  69  t h e y should be p l a c e d i n the same genus.  Many congeneric  s p e c i e s , however, show t o t a l h y b r i d s t e r i l i t y ,  and f a r l e s s  m o r p h o l o g i c a l and e c o l o g i c a l d i v e r g e n c e than do A c r o c h e i l u s and P t y c h o c h e i l u s . genera?  Should these be p l a c e d i n separate  I f the c r i t e r i o n f o r taxonomic rank were t o be  recentness o f divergence from a common ancestor, t h e r e i s no d i r e c t evidence which i s r e l e v a n t t o the problem a t hand. In f a c t ,  g e n e t i c divergence, or most commonly, m o r p h o l o g i c a l  divergence,  i s used as an i n d i c a t o r o f the time o f d i v e r g e n c e  from a common a n c e s t o r .  The m o r p h o l o g i c a l and g e n e t i c  evidence, however, are o f t e n i n c o n f l i c t f o r the same s p e c i e s pair.  A f u r t h e r problem i n reassessment  o f the g e n e r i c  s t a t u s o f A c r o c h e i l u s and P t y c h o c h e i l u s i s t h a t t h e r e are not s u f f i c i e n t data a v a i l a b l e on t h e i r degree o f r e l a t i o n s h i p t o the many o t h e r genera o f western  c y p r i n i d s not  considered here. I f A c r o c h e i l u s and P t y c h o c h e i l u s were p l a c e d i n the same genus, 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 should p r o b a b l y a l s o be p l a c e d w i t h them.  However, s i n c e the r e l a t i o n s h i p  o f these f o u r t o o t h e r genera  i s unknown, the b e s t p o s i t i o n  to adopt seems t o be to r e t a i n the p r e s e n t u n t i l the r e q u i r e d data are a v a i l a b l e .  classification  SUMMARY The w i l d i n t e r m e d i a t e s Ptychocheilus  b e t w e e n A c r o c h e i l u s and  are of hybrid o r i g i n ,  and most  of  them a r e F-^ h y b r i d s . When r e l a t i v e is  abundance o f  disproportionate,  interspecific  in  conditions  None o f  collected  caused b y d r i f t i n g  are  i n Wolfe unaltered  gametes  chance.  the m o r p h o l o g i c a l evidence  swamping o r rare,  than M i s s e z u l a Lake  evidence  is  A l l known  t h a t h y b r i d i z a t i o n under  may b e  meeting by  that habitat modification  other  and t h e  Creek suggests  active  place.  to h y b r i d i z a t i o n .  localities  streams,  i n M i s s e z u l a Lake,  species  r a r e F ^ h y b r i d s f r o m many o t h e r  suggests  not p r e r e q u i s i t e hybrid  two p a r e n t a l  mating probably takes  The o c c u r r e n c e o f localities  as  the  i n t r o g r e s s i o n has  suggests  taken p l a c e ,  aberrant i n d i v i d u a l s are almost  that although  certainly  backcrosses. The mechanisms w h i c h h a v e p r e v e n t e d between the p a r e n t a l sterility  i n the  gene  species are a high degree  F^ h y b r i d s ,  of h y b r i d s , which lowers  the numerical  against  recombinants  of  rarity  the p r o b a b i l i t y o f  to h y b r i d mating or of backcrossing, severe selection  flow  hybrid  and p r o b a b l e in  any  71 backcrosses 6.  which are produced.  Only i n M i s s e z u l a Lake i s high  enough,  parental  relative  species,  to  to  the the  rate of h y b r i d i z a t i o n abundance o f  suggest that  t h r o u g h h y b r i d i z a t i o n may a f f e c t size of  7.  a parental species.  hybrid  localities,  levels  of  and h y b r i d s c o n s t i t u t e  of  The p r e s e n c e  o f dominance e f f e c t s  presence  similar.  f e r t i l i t y of  i n the  1.3  per  inheritance  combined w i t h  the  the h y b r i d s ,  the  i n chromosome m o r p h o l o g y ,  of backcrosses  Acrocheilus  high  the p a r e n t a l p o p u l a t i o n s .  some p a r e n t a l c h a r a c t e r s ,  similarity  population  b o t h p a r e n t a l s p e c i e s have  abundance,  observed p a r t i a l  the  loss  I n t h e o t h e r known  cent or l e s s  of  gamete  either  and  the  i n the w i l d suggest  and P t y c h o c h e i l u s  that  are g e n e t i c a l l y  very  72 LITERATURE CITED  A n d e r s o n , E . 1949. New Y o r k : i x +  Introgressive hybridization. 109.  1953. Introgressive hybridization. 280-307.  J.  Biol.  Rev.  Bigelow, R.S. 1965. H y b r i d z o n e s and r e p r o d u c t i v e E v o l u t i o n , L a n c a s t e r , P a . 19: 4 4 9 - 4 5 8 . D a r l i n g t o n , C D . 1958. Evolution of & B o y d , E d i n b u r g h : x i i + 265.  genetic  Wiley,  28: isolation.  systems.  E p l i n g , C a r l 1947. Genetic aspects of natural A c t u a l and p o t e n t i a l gene f l o w i n n a t u r a l Am. N a t . 8 1 : 1 0 4 - 1 1 3 .  Oliver  populations. populations.  G i l b e r t , C a r t e r R. 1961. Hybridization versus intergradation: a n i n q u i r y i n t o t h e r e l a t i o n s h i p o f two c y p r i n i d f i s h e s . C o p e i a , 1961: 181-192. Hubbs, C a r l L . 1955. H y b r i d i z a t i o n between f i s h nature. S y s t . Z o o l . 4: 1-20.  species  in  Hubbs, C a r l L . and L . C . H u b b s , 1 9 3 2 a . Apparent parthenog e n e s i s i n n a t u r e i n a form o f f i s h o f h y b r i d o r i g i n . Science, N.Y. 76: 6 2 8 - 6 3 0 . 1932b. Experimental v e r i f i c a t i o n of natural h y b r i d i z a t i o n between d i s t i n c t genera o f s u n f i s h e s . P a p . M i c h . A c a d . S c i . 15: 4 2 7 - 4 3 7 . 1933. The i n c r e a s e d g r o w t h , p r e d o m i n a n t m a l e n e s s , and apparent i n f e r t i l i t y of h y b r i d sunfishes. Pap. M i c h . A c a d . S c i . 17: 6 1 3 - 6 4 1 . Hubbs, C a r l L . , L . C . Hubbs and Raymond E . J o h n s o n , 1943. H y b r i d i z a t i o n i n n a t u r e between s p e c i e s o f s u c k e r s . C o n t r . L a b . V e r t e b r . B i o l . - U n i v . M i c h . 22: 1-76. H u b b s , C a r l L . and K . F . L a g l e r , 1947. Lakes Region. B u l l . Cranb. Inst.  F i s h e s o f the Great S c i . 26: 1 - 1 8 6 .  Hubbs, C a r l L . and L . P . S c h u l t z , o f the Columbia R i v e r chub. M i c h . 232: 1-7.  The s c i e n t i f i c Pap. Mus. Z o o l .  1931. Occ.  name Univ.  73  H u b b s , C a r l L . , B o y d W. W a l k e r and Raymond E . J o h n s o n , 1943. H y b r i d i z a t i o n i n n a t u r e between s p e c i e s o f American cyprinodont fishes. Contr. Lab. Vertebr. B i o l . Univ. M i c h . 23: 1-21. H u b b s , C l a r k , 1957. D u r a t i o n o f sperm v i a b i l i t y as a f a c t o r i n frequency of f i s h - h y b r i d i z a t i o n . T e x . J . S c i . 9: 472-474. 1958. F e r t i l i t y o f F-j_hybrids between the p e r c i d f i s h e s , E t h e o s t o m a s p e c t a b i l e and E . l e p i d u m . Copeia, 1958: 5 7 - 5 9 . 1959. L a b o r a t o r y h y b r i d c o m b i n a t i o n s among etheostoraatine f i s h e s . T e x . J . S c i . 11: 4 9 - 5 6 . 1960. D u r a t i o n o f sperm f u n c t i o n i n t h e p e r c i d f i s h e s E t h e o s t o m a l e p i d u m and _E. s p e c t a b i l e , a s s o c i a t e d w i t h sympatry o f the p a r e n t a l p o p u l a t i o n s . C o p e i a , 1960: 1-8. 1961. D i f f e r e n c e s b e t w e e n t h e gamete c o m p a t i b i l i t y o f E t h e o s t o m a l e p i d u m sperm and eggs o f r e l a t e d p e r c i d f i s h e s f r o m a l l o p a t r i c and s y m p a t r i c p o p u l a t i o n s . Tex. J . S c i . 13: 427-433. Mayr,  E r n s t , 1963. A n i m a l s p e c i e s and e v o l u t i o n . Belknap P r e s s , H a r v a r d U n i v e r s i t y P r e s s , Cambridge: x i v + 797.  Parker, B.  Raymond C . , 1950. H o e b e r , New Y o r k :  Methods o f xv + 294.  tissue culture.  Paul  P a t t e n , B e n j a m i n G . , 1960. A h i g h i n c i d e n c e o f the h y b r i d A c r o c h e i l u s "alutaceum x P t y c h o c h e i l u s o r e g o n e n s e . 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Descriptions of new i n t e r g e n e r i c h y b r i d s b e t w e e n c e r t a i n c y p r i n i d f i s h e s o f the northwestern U n i t e d S t a t e s . Proc. B i o l . Soc. Wash. 49: 1-10. S i n n o t t , Edmund W., L . C . Dunn and T . D o b z h a n s k y , 1958. P r i n c i p l e s Of g e n e t i c s . M c G r a w - H i l l , New Y o r k : x i v + 459. S n y d e r , J o h n O t t e r b e i n , 1905. Critical l a t e r a l i s and L e u c i s c u s c a u r i n u s .  Fish.  1904:  n o t e s on M y l o c h e i l u s R e p t . U . S . Comranr.  341-342.  1908. F i s h e s of the Northern C a l i f o r n i a . 153-159.  c o a s t a l s t r e a m s o f O r e g o n and B u l l . B u r . F i s h . Wash. 27, 1907:  T o w n s l e y , P . M . , H . G . W r i g h t and M . A . S c o t t , 1963. fish tissue culture. J . F i s h . Res. B d . Can. 679-684.  Marine 20:  W e i s e l , G e o r g e F . , 1954. A r e d i s c o v e r e d c y p r i n i d h y b r i d from Western Montana, M y l o c h e i l u s caurinum x R i c h a r d s o n i u s balteatus balteatus. C o p e i a , 1954: 2 7 8 - 2 8 2 . 1 9 5 5 a . The o s t e o l o g y o f M y l o c h e i l u s c a u r i n u m x P t y c h o c h e i l u s o r e g o n e n s e , a c y p r i n i d h y b r i d , compared with its parental species. J . M o r p h . 96: 3 3 3 - 3 5 8 . 1955b. T h r e e new i n t e r g e n e r i c f i s h e s from Western Montana, 396-411.  hybrids of cyprinid Am. M i d i . N a t . 53:  W o l f e , K . and C . E . D u n b a r , 1958. An e x p l a n a t i o n o f t h e p r i n c i p l e s and methods o f t i s s u e c u l t u r e . Progve. F i s h C u l t . 20: 3-7. W o l f e , K . , M . C . Quimby, E . A . P y l e and R . P . D e x t e r , 1960. P r e p a r a t i o n o f monolayer c e l l c u l t u r e s from t i s s u e s o f some l o w e r v e r t e b r a t e s . S c i e n c e N . Y . 132: 1890-1891.  

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