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An examination of two reported protein polymorphisms in threespine stickleback, Gasterosteus aculeatus Michiel, Glen Patrick 1978

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AN EXAMINATION  OF TWO REPORTED PROTEIN  POLYMORPHISMS IN THREESPINE STICKLEBACK, GASTEROSTEUS  ACULEATUS  by GLEN PATRICK MICHIEL B.Sc.  (Hons.), C a r l e t o n  University,  1974  THESIS SUBMITTED IN PARTIAL FULFILLMENT THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE FACULTY OF GRADUATE STUDIES Department of Zoology  we accept t h i s t h e s i s as conforming to the r e q u i r e d standard  THE UNIVERSITY OF BRITISH COLUMBIA April,  1977  Glen Patrick M i c h i e l , 1978  In  presenting this  thesis  an advanced degree at the L i b r a r y s h a l l I  f u r t h e r agree  in p a r t i a l  fulfilment of  the requirements f o r  the U n i v e r s i t y of B r i t i s h Columbia,  make i t  freely available  that permission  for  I agree  r e f e r e n c e and  f o r e x t e n s i v e copying o f  this  that  study. thesis  f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s of  this  representatives. thesis  It  is understood that copying or p u b l i c a t i o n  f o r f i n a n c i a l gain s h a l l  not be allowed without my  w r i t ten pe rm i ss i on .  G. P. MICEIF.L  ZOOLOGY. Department of The U n i v e r s i t y o f B r i t i s h 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  Date  QCT.  lk . 77  Columbia  i  Abstract  Gasterosteus  aculeatus  is  i n morphological  It  i s found  northern found  t e r m s , an  i n a wide v a r i e t y  hemisphere.  Forms o f  i n anadromous and  examinations concerning stickleback Two ported  the  extremely  variable species.  threespine  freshwater  situations.  the d i s t r i b u t i o n  the  s t i c k l e b a c k are Taxonomic  of various  forms  i n the  literature.  i n south  A hemoglobin polymorphism  E u r o p e a n s t i c k l e b a c k , and (enzyme:  of a l l e l e s  i n both  to hypothesize  these  In t h i s  study  I t e s t e d these  southwestern B r i t i s h  habitat  types.  apparently., provided  the  The  caused the  has  One  a relict  researchers  hypotheses with across  selective pressure  to t e s t  to  the  populations  a range of  macro-  s t i c k l e b a c k s sampled i s ,  anadromous p o p u l a t i o n .  opportunity  been  forms of s t i c k l e b a c k .  Columbia,  p o p u l a t i o n of  has  distribution  t h a t t h e p o l y m o r p h i s m s were r e l a t e d  anadromous e x i s t e n c e s o f c e r t a i n  re-  a muscle  c r e a t i n e kinase)  situations  of  results.  i n s t i c k l e b a c k f r o m w e s t e r n Canada.  after)  stickleback  of h a b i t a t s throughout  have p r o d u c e d e q u i v o c a l  myogen p o l y m o r p h i s m  from  threespine  s t i c k l e b a c k p r o t e i n p o l y m o r p h i s m s have b e e n  been found  found  L.,  This  population  a population during  to adapt to a freshwater  (or  existence.  No e v i d e n c e o f t h e h e m o g l o b i n p o l y m o r p h i s m was indeed  some d o u b t was  c a s t upon t h e E u r o p e a n work due t o  technique aberrations.  The m u s c l e myogen p o l y m o r p h i s m  present  i n a few p o p u l a t i o n s ,  pattern  emerged.  selectively  Electrophoretic myogen p a t t e r n s  of these a l l e l e s  k i n a s e ) was  patterns  o f t h e h e m o g l o b i n and m u s c l e  provoked the suggestion  are  the  reported  must be r e j e c t e d .  suggested.  various  to a single  f o u n d t o be i n c o n s i s t e n t w i t h t h e  The h y p o t h e s i s r e l a t i n g existences  that  o f t h e m u s c l e myogen p o l y m o r p h i s m  structure generally  being  i s discussed.  f o r m s o f s t i c k l e b a c k do i n d e e d b e l o n g The p a t t e r n  was  however, no b a s i c e n v i r o n m e n t a l  The p o s s i b i l i t y  neutral  found,  species. (creatine  dimeric  f o r t h e enzyme. the polymorphisms Several  t o anadromous  alternative projects  iii  Table  o f Contents  Abstract Table  i  o f Contents  List  of Tables  List  of Figures  i i i iv v  Acknowledgements  vi  CHAPTER I . GENERAL INTRODUCTION CHAPTER I I . STICKLEBACK HEMOGLOBIN  1 11  1.  Introduction  11  2.  Materials  15  3.  Results  4.  Discussion  and Methods  20 and C o n c l u s i o n s  CHAPTER I I I . STICKLEBACK MUSCLE MYOGEN  25 27  1.  Introduction  27  2.  Materials  34  3.  Results  4.  Discussion  and Methods  39 and C o n c l u s i o n s  43  CHAPTER I V . GENERAL DISCUSSION  47  LITERATURE CITED  58  iv  L I S T OF TABLES  TABLE  I . Summary  o f hemoglobin  sample  l o c a t i o n s and  results TABLE  I I . A summary  21 o f m u s c l e myogen p o p u l a t i o n  locat-  i o n s , h a b i t a t and f o r m TABLE I I I . A summary o f V a n c o u v e r sampled TABLE TABLE  33 Island  populations  .  I V . M u s c l e myogen g e n o t y p e  38 frequencies  40  V. A c o m p a r i s o n o f s i x s p e c i e s d e m o n s t r a t i n g a s i m i l a r m u s c l e myogen p r o t e i n p o l y m o r p h i s m  51  V  List  of Figures  FIGURE 1.  Three forms o f Gasterosteus. a c u l e a t u s  FIGURE 2.  Patterns  o f the Reported Hemoglobin  Poly-  morphism Found i n I t a l y FIGURE  3.  Geographic Locations  ulations  12 o f S t i c k l e b a c k Pop-  Sampled f o r H e m o g l o b i n P o l y m o r p h i s m  FIGURE 4.  The A b e r r a n t  FIGURE 5.  The M u s c l e Myogen P a t t e r n  FIGURE 6.  Geographic Locations  ulations  Sampled  Gel  7.  Age and S e a s o n  FIGURE  8.  Geographic Locations  28  o f S t i c k l e b a c k Pop-  nary  Hypothetical Pattern  36 41  of the Polymorphic  Populations FIGURE 9.  17 22  f o r t h e Myogen P o l y m o r p h i s m  FIGURE  3  44 f o r Dimeric  Quarter-  S t r u c t u r e o f t h e Enzyme C r e a t i n e K i n a s e  51  vi  Acknowledgements Help comes i n strange advice  forms, t o a graduate  student,  (hard t o t a k e ) , "know-how", p a t i e n c e , funds,  con-  v e r s a t i o n and most i m p o r t a n t l y c r i t i c i s m  ( a l s o hard t o  t a k e ) . I t i s obvious  t o the many who  helped me, i s very  to me t h a t my debt,  great.  I would p a r t i c u l a r l y l i k e to thank my a d v i s o r J.D. McPhail,  f o r h i s encouragement, "penelopean" p a t i e n c e and  advice d u r i n g my s t u d i e s . thank Dr. McPhail me as a student call  I would a l s o p a r t i c u l a r l y l i k e to  f o r t a k i n g the i n i t i a l gamble of a c c e p t i n g  (when o t h e r s had r e f u s e d ) .  He would  probably  i t "succumbing to a study i n p e r s i s t a n c e " . I would a l s o l i k e t o thank the other members o f my  committee, Dr. B i l l N e i l l , and Dr. Judy Myers. c r i t i c i s e d my work and t h e s i s d r a f t s .  Both k i n d l y  Dr. Myers a l s o s u p p l i e d  me with l a b space. The  f o l l o w i n g people  c o n t r i b u t e d hours o f help d u r i n g  my f i e l d , head and l a b work, E. Black, D. B e l f o r d , M. Karen, C. Murray, A. Peacock, D. Peacock  (no r e l a t i o n ) , P. P r e s s l e y ,  A. Robertson, G. R o s s i t e r (my t y p i s t ) and t h a t phenomenon J . Spence. experience.  unusual  Thank you a l l , f o r a most i n t e r e s t i n g  vii  Utram b i b i s ?  Aquam an undam? o r i g i n - unknown  1  CHAPTER I.' GENERAL INTRODUCTION Much s c i e n t i f i c a t t e n t i o n has been d i r e c t e d towards m o r p h o l o g i c a l v a r i a t i o n i n the s p e c i e s complex  Gasterosteus  a c u l e a t u s , the t h r e e s p i n e s t i c k l e b a c k . M o r p h o l o g i c a l f e a t u r e s common to t h i s f i s h such  as  p l a t e number, spine number, spine l e n g t h , mating c o l o u r , s i z e , and v e r t e b r a l number have been demonstrated to be extremely v a r i a b l e over a range of l o c a t i o n s ,  (Hagen, 19 67;  M c P h a i l , 1969; McPhail and Lindsey, 1970; Hagen and G i l b e r t s o n , 1972) .  F i s h w i t h c e r t a i n combinations  of these m o r p h o l o g i c a l  f e a t u r e s have been given names by taxonomists, w i t h C u v i e r and V a l e n c i e n n e s ,  beginning  (1829), ( c i t e d i n Munzing,  1963,) f o r the sake of d e s c r i p t i v e convenience.  These forms  of Gasterosteus a c u l e a t u s are d e s c r i b e d as l e i u r u s , armatus and t r a c h u r u s , ( F i g . 1 ) .  semi-  L e i u r u s i s the unarmoured  extreme, t r a c h u r u s i s the w e l l armoured extreme, and armatus i s a moderate form between the two extremes.  semiThere  has been some d i s c u s s i o n with regard to the p h y l o g e n e t i c r e l a t i o n s h i p s among these forms,  (Hagen, 1967; M i l l e r  and  Hubbs, 1969; Hagen and M c P h a i l , 1970), however no consensus has been reached.  One  t h i n g i s c l e a r from t h i s c o n t r o v e r s y :  s t i c k l e b a c k are not a t y p i c a l s p e c i e s i n the sense.  traditional  I t should be noted t h a t the forms do not  satisfy  Mayr's (1963) c r i t e r i o n f o r s u b - s p e c i e s , as the p o p u l a t i o n s  2  in British  Columbia are not  With the the  r e l a t i o n s h i p s of  w o r k e r s have a p p l i e d t h e  1967; it  of  Cucchi,  1969;  and  the  Jones, unpublished).  Aspinwall, with  (1967) r e p o r t e d later  e t al.,1968) stated  identified  i n the  between f r e s h w a t e r populations two  i n the  a t the  as  (trachurus)  freshwater  and  malate  dehydrogenase  of  loci  were  Two  poly-  t h e m u s c l e myogen  creatine kinase  (Gosselin-Rey  v a r i a b l e and  c o u l d be  used to  (leiurus),  further  distinguish  and  anadromous  i n l o c a t i o n s where t h e r a n g e s o f  Callegarini  and  Cucchi  (1968)  a hemoglobin polymorphism which a l s o appeared with  work  literature.  t h a t one  populations  phases abutted.  From t h i s  Several other  s t i c k l e b a c k was  t h a t the v a r i a b i l i t y  comm.; Hagen,  inconclusive results.  morphisms have been r e p o r t e d  proteins,  invariable  comm.).  some o f  of e l e c t r o p h o r e s i s to  alpha-glycerophosphate  (Aspinwall, pers.  Hagen  isolated.  species, several  stickleback, (Aspinwall, pers.  d e h y d r o g e n a s e and  examined by  the  technique  appears t h a t s t i c k l e b a c k are  loci,  geographically  hope t h a t e n z y m a t i c work w o u l d c l a r i f y  phylogenetic  populations  clearly  anadromous e x i s t e n c e s , i n  the  reported  to c o r r e l a t e southern  Europe. Although populations aculeatus  a definitive  and  has  c o m p a r i s o n between E u r o p e a n  North American p o p u l a t i o n s  not  been c a r r i e d  out  of  i t should  the European s t i c k l e b a c k s e l e c t r o p h o r e t i c a l l y  Gasterosteus be  noted  studied  that by  3  FIGURE I Three Forms of Gasterosteus Aculeatus These three forms of Gasterosteus have been given names by taxonomists for descriptive convenience. Form A i s c a l l e d l e i u r u s , B semi-armatus and C trachurus. Often t h i s nomenclature i s based upon the number of l a t e r a l armour plates; however there i s a spectrum of forms between the extremes and the d i s t i n c t i o n s are often a r b i t r a r y . (Adapted from, McPhail and Lindsey, 1970, and Hagen and Gilbertson, 1972)  5  Italian  r e s e a r c h e r s , resemble m o r p h o l o g i c a l l y , the North  American found and  leiurus  along  form.  In Europe, the trachurus  the northern  i n the Black  coast  Sea a r e a .  found  i n southwestern  etc.)  (Munzing,  ( S c o t l a n d , Norway, U.S.S.R)  The l e i u r u s  Europe  arguments  variability  (Lewontin,  the n e o d a r w i n i s t  nearly  controversy  over  197 4 ) , i t was i n t e r e s t i n g  view.  for this  studies i s irrelevant The n e o d a r w i n i s t ' s  t o note  thereby  individuals An  examination  alternatives these  they  to the w e l l being  (or selectionist's)  are i n d i v i d u a l l y  of the l i t e r a t u r e  way t h a t t h e s e possibilities follows:  of the view i s t h a t  selected units  provides  forvariability  u t i l i t y to  Obviously  several  patterns.  do n o t n e c e s s a r i l y r e f l e c t  f e r e n c e s between i n d i v i d u a l s . with phylogenetic  view i s  a r e found i n .  accounting  alternatives  both  r e v e a l e d by e l e c t r o -  o f h e r e d i t y , t h a t s u r v i v e on t h e b a s i s o f t h e i r the  that  supporting  the n e u t r a l i s t  variability  a l l electroisozymes  t h e meaning  s t i c k l e b a c k were  adaptive,  Basically,  t h a t much o f t h e e n z y m a t i c  organism.  Albania,  and t h e n e u t r a l i s t / n e o d a r w i n i s t  t o be e n v i r o n m e n t a l l y  phoretic  Italy,  1963).  t h e o n l y known p o l y m o r p h i s m s thought  form i s g e n e r a l l y  (Spain, France,  Considering the unresolved of biochemical  form i s  then,  genetic  accounting  factors  dif-  any r e s e a r c h  i m p l i c a t i o n s must be p e r f o r m e d  confounding  Some o f  i n such  are neutralized.  forprotein variability  Some  a r e as  a  6  (1)  Fish  Age  S e v e r a l g r o u p s o f r e s e a r c h e r s have r e p o r t e d changes i n f i s h  hemoglobin.  W i l k i n s and  lies  example, have f o u n d marked c h a n g e s i n t h e components o f h e r r i n g situations exist  e t a l . , 1964) , t h e r o a c h erythrophthalmus) eelpout for to  (Rutilus  ( P e r e z and  dent  on  age.  In f i s h ,  s h i p between l e n g t h and  age  and  Seasonal  variability  Yousef  and  (1975),  appeared  t o be  possible  there i s usually  age a  does  relation-  have been n o t e d  hemoglobin, by  Denton  (Salmo g a i r d n e r i ) .  place independently  a d a p t a t i o n s and  activity  in  of temperature  to other environmental  and  Interest-  discovered that this variation  i n response  depen-  Variability  i n rainbow t r o u t  Diet, metabolic  compensate variability  i n concentrations of  serum p r o t e i n  enough, i t was  (Scardinius  i n the  To of  (Koch  (within populations).  Temperature  c o n c e n t r a t i o n s took  and  factors.  were s u g g e s t e d  as  explanations.  Baldwin trout  and  determination of r e l a t i v e  Seasonal  hematocrit,  ingly  the rudd  of polymorphisms i s not  n o t p r e s e n t a g r e a t p r o b l e m , as  (2)  1974)  must examine c a s e s  t h a t the d i s t r i b u t i o n  Similar  (Salmo s a l a r )  rutilus),  Maclean,  age.  (Zoarus v i v i p a r u s ) ( H j o r t h , 1974).  t h i s phenomenon, one see  salmon  (1966) f o r  electrophoretic  (Clupea harengus) w i t h  i n the A t l a n t i c  ontogenetic  and  Hochachka  (1970) have shown t h a t r a i n b o w  (Salmo ' g a i r d n e r i ) a c c l i m a t e d t o d i f f e r e n t  temperature  7  regimes e x h i b i t d i f f e r e n t acetylcholinesterase.  electrophoretic patterns  This  of brain  i s interesting since, i n v i t r o ,  many enzymes have n a r r o w t e m p e r a t u r e optimums where efficiency (3)  their  i s a t a maximum.  Environmental  Adaptation  A most i n t e r e s t i n g example o f a p p a r e n t e l e c t r o p h o r e t i c allozyme adaptation strated insignis ability  that  two s y m p a t r i c  the f i s h  found  are very  Catostomis  micro h a b i t a t  similar,  C. i n s i g n i s  b u t C. c l a r k i i i s  s e c t i o n s o f streams.  component a l l o w e d  spite of increased  t o be a d v a n t a g e o u s t o  Powers t h e o r i z e d  tissue acidity  Biochemical  .One w o u l d t h i n k  have t h i s  pH i n -  that the extra  t o remain i n the f a s t  some o x y g e n s u p p l y  thereby avoiding  C. i n s i g n i s .  considered  C. c l a r k i i  w a t e r by m a i n t a i n i n g  component  components a r e a l l f u n c t -  So, why d o e s C. c l a r k i i  d e p e n d e n t component?  adaptation.  T h i s dependence i s termed t h e  and i s n o r m a l l y  organism.  vari-  characterized i sfunctionally  d e p e n d e n t upon pH.  Bohr e f f e c t  theory.  fish,  He demon-  h a s an e x t r a e l e c t r o p h o r e t i c h e m o g l o b i n  i n d e p e n d e n t o f pH,.  action,  (1972).  e x h i b i t hemoglobin  i n faster flowing  w h i c h when b i o c h e m i c a l l y  the  catostomid  t h a t appears t o c o i n c i d e with  C. c l a r k i i  ionally  by Powers,  and C a t o s t o m i s c l a r k i i  Externally normally  was c i t e d  direct  flowing  t o muscle t i s s u e , i n  c a u s e d by t h e e x t r a  spatial  competition  with  evidence appears t o support that  living  muscle  i n faster flowing  this water  8  would i n v o l v e more muscular a c t i v i t y , thereby producing more l a c t i c acid. subjected  Black  (1958) has  shown t h a t when f i s h e s  are  to extreme e x e r t i o n the b u i l d up of l a c t i c a c i d i n  the blood can be g r e a t enough to overwhelm the b u f f e r of the blood and  produce a pH  change.  capacity  Black a l s o showed  t h a t t h i s pH  change l a s t e d f o r f a i r l y  long p e r i o d s  (24 h o u r s ) .  Thus i t would appear t h a t a pH  of time  independent  component would be extremely u s e f u l to an organism under these c i r c u m s t a n c e s . (4)  Tissue An  Variability  i n t e r e s t i n g pattern  of p r o t e i n v a r i a t i o n i n v o l v e s  v a r i a b i l i t y among t i s s u e s w i t h i n an i n d i v i d u a l organism. S c h o l l and  Eppenberger,  (1972) d i s c u s s e d  the t i s s u e v a r i -  a b i l i t y of c r e a t i n e kinase i n a v a r i e t y of f i s h Other r e s e a r c h e r s respect birds  have i l l u s t r a t e d the  to c r e a t i n e k i n a s e i n f r o g s  species.  same phenomenon w i t h  ( L y s l o v a , 1971)  and  (Eppenberger, 1968). Examining a s p e c i f i c type of t i s s u e w i t h i n  of a p o p u l a t i o n  can  overcome c o n f u s i o n  individuals  generated by t h i s  phenomenon. (5)  Historical  Situations  Although i t i s a d i f f i c u l t concept to several researchers  have attempted to show d i f f e r e n c e s based  upon h i s t o r i c a l s e p a r a t i o n s Aspinwall  (1974) has  between odd  and  illustrate,  (neutral  drift).  i l l u s t r a t e d gene frequency  differences  even year c l a s s e s of c y c l i n g pink salmon,  9  Oncorhyrchus gorbuscha. study  because the year  demes. is  Pink  salmon were i d e a l  classes represent  T h e r e i s no m i g r a t i o n  assumed t h a t t h e  two  between t h e s e  by  historical  assumed t h a t t h e  selective  accident.  f o r c e s on  Most i m p o r t a n t l y ,  m o d e r a t e amount o f m i g r a t i o n  the v a r i a b i l i t y  one,  and  also  class  between c o a s t a l r i v e r s  w i t h i n a year  over  a wide  have  that  variability  It  have  Aspinwall  illustrated  g r e a t enough t o keep t h e between y e a r than  individual  each year  he  this  populations.  demes were f o r m e r l y  become s e p a r a t e d  been c o n s t a n t .  two  for  the  was  greater  geographical  range. Now,  f o r the  stickleback, with  two  both  r e p o r t e d p r o t e i n polymorphisms i n  polymorphisms are  anadromous e x i s t e n c e s .  saltwater populations Columbia,, an In B r i t i s h of the  ideal  i n freshwater  t h a t by  and  designing  of morphological and  gross  division  i n Europe.  s a l t w a t e r , however t h i s I felt  exists  s t i c k l e b a c k are  v i o u s l y mentioned found  the  the  freshwater  the  one  to t e s t  these  between t h e less  clear  Normally trachurus  distinction  variation,  h a b i t a t types  both  and  a v a i l a b l e i n southwestern  situation  Columbia  forms o f  are  Since  reportedly associated  the  or  hypotheses.  distributions  than  those  leiurus  pre-  form i s  form i s found  in  i s a gross g e n e r a l i z a t i o n .  samples t o c o v e r salt  British  freshwater  c o u l d get  the  spectrum  existences,  a c l e a r p i c t u r e of  distribution  of the polymorphisms w i t h i n a r e l a t i v e l y  geographical  area.  the  small  10  B e c a u s e t h e r e were d i f f e r e n c e s histories decided two  o f t h e two  t o r e p o r t my  individual  i n the techniques  r e p o r t e d p r o t e i n polymorphisms examination  I have  o f them i n d e p e n d e n t l y i n  chapters each w i t h b r i e f  f o l l o w e d by a f i n a l  and  chapter of general  specific conclusions, discussion.  11  CHAPTER I I .  1.  STICKLEBACK HEMOGLOBIN  Introduction I t a l i a n researchers  ( C a l l e g a r i n i and C u c c h i ,  C a l l e g a r i n i and C u c c h i , 1969; 1969;  and Raunich e t a l . ,  C u c c h i , 1969;  1972)  1968;  Raunich e t a l . ,  have examined a hemoglobin  polymorphism i n European s t i c k l e b a c k . T h i s polymorphism i n v o l v e s three phenotypes A, AB and (Fig.2).  Phenotype A i s found i n non-migratory  freshwater  p o p u l a t i o n s of s t i c k l e b a c k from western I t a l y and Germany..  B  southern  P o p u l a t i o n s from the v i c i n i t y of Verona are a l s o  monomorphic f o r phenotype A.  Phenotypes AB  become more common i n p o p u l a t i o n s as one  and B then  approaches the  A d r i a t i c Sea, p a r t i c u l a r l y i n b r a c k i s h water s i t u a t i o n s . U l t i m a t e l y c o a s t a l p o p u l a t i o n s around Venice and Ravena have a g r e a t e r frequency of the Hb Raunich e t a l . , the. H  a l l e l e was  allele  (1972) generated  than the Hb  by the migratory  A  allele.  the hypothesis t h a t  r e l a t e d to the b r a c k i s h and  i s t e n c e encountered suggested  B  s a l t water  sticklebacks.  t h a t r e s e a r c h e r s from other c o a s t a l areas  They should  i n v e s t i g a t e t h i s hypothesis to see i f the polymorphism  was  present i n o t h e r m i g r a t o r y p o p u l a t i o n s . S e v e r a l q u e s t i o n s arose when I examined t h i s work. F i r s t l y , was  ex-  the polymorphism a g e n e t i c phenomenon?  FIGURE 2 P a t t e r n s o f t h e Reported Hemoglobin In I t a l y  Polymorphism  The component p a t t e r n s found i n I t a l y a r e t y p i f i e d by 3 phenotypes, A,AB,and B.These phenotypes s e p a r a t e i n t o d i f f e r e n t component p a t t e r n s a t d i f f e r e n t pH v a l u e s . A l l bands a r e anodal,S i s the s t a r t i n g p o i n t . B r i t i s h Columbia s t i c k l e b a c k a r e t y p i f i e d by t h e A p a t t e r n i n diagram ( a ) , ( R a u n i c h e t a l . , 1 9 7 2 ) .  13  +  (a) p H 8 . 6  A  AB  B  (b) p H 3.5  A  AB  B  14  The  Italians  suggested  experiments,  t h a t i t was.  the results  a l l e l e was t h e p r o d u c t  o f which  ratios  e v e n have t o be e m p l o y e d  other  people's  breeding  (McPhail,  survivorship with  breeding  t h a t t h e Hb Their figures  that i n fact  (an a m a z i n g  lack of success  experiment  suggested  a s i n g l e mutation.  were so c l o s e t o e x p e c t e d not  They c o n d u c t e d  fact  statistics  did  considering  pers.  comm.) a t  western North  American  stickleback). Secondly,  they  suggested  that the presence o f the  polymorphism i s r e l a t e d  t o t h e b r a c k i s h and s a l t  existences of migratory  populations.  arises, and  why w e r e n ' t p o p u l a t i o n s  Naples polymorphic?  morphological migratory  water  The q u e s t i o n  from t h e v i c i n i t y  The I t a l i a n  then o f Rome  work s t a t e s t h a t  f e a t u r e s were u s e d t o d i s t i n g u i s h  and s t a t i o n a r y p o p u l a t i o n s ,  between  a n d t h e Rome and  Naples populations  apparently  criterion.  d i s t i n g u i s h i n g f e a t u r e s were n o t s p e c i f i e d .  The  These  third  Europeans pose. coastal  for  of stickleback i n  t h i s p r o t e i n polymorphism?  i n southwestern B r i t i s h  situation,  i s the question the  populations  exhibit  Columbia occupy a s i m i l a r  t h e r e f o r e , I s e t o u t t o examine  t h e presence o f t h e hemoglobin polymorphism.  p o l y m o r p h i s m was p r e s e n t on  that arises  Do o t h e r  situations  Stickleback coastal  question  f i t the stationary population  a micro-geographic  that  I planned  I f the  t o examine i t s d i s t r i b u t i o n  scale to test  t h e p o l y m o r p h i s m was r e l a t e d  populations  the I t a l i a n  hypothesis  t o a b r a c k i s h water  existence.  15  2.  M a t e r i a l s and  (a)  Field The  Methods  Techniques  s t i c k l e b a c k were c o l l e c t e d with beach s e i n e s ,  s e i n e s and minnow t r a p s .  The  f i s h were r e t u r n e d  pole  to the  lab  and  kept a l i v e u n t i l immediately b e f o r e the  electrophoresis  was  to b e g i n .  extremely  The  M o r t a l i t y during  f i s h were c o l d shocked then k i l l e d .  extracted  4%  i n t o the bulbous a r t e r i o s u s .  drawn i n t o a p a s t e u r p i p e t c o n t a i n i n g (w/v)  sodium c i t r a t e  as an a n t i c o a g u l a n t .  solution.  The  blood  separate the e r y t h r o c y t e s . three was  Blood  times a t 0°C  replaced  The  was  anBlood  a s m a l l amount of  sodium c i t r a t e  then c e n t r i f u g e d  This operation  at about 5,000 r.p.m.  a f t e r each c e n t r i f u g a t i o n .  low.  was  (Koehn, 196 9) by making an i n c i s i o n i n the  t e r i o r v e n t r a l region was  storage was  was The  The  acted to  performed supernatant erythrocytes  were then l y s e d by adding a s m a l l volume of d i s t i l l e d water to the p l u g of packed c e l l s . c e n t r i f u g e d out  (b)  The  c e l l p a r t i c l e s were  l e a v i n g the hemoglobin i n the  again  supernatant.  E l e c t r o p h o r e t i c Techniques E l e c t r o p h o r e s i s was  c a r r i e d out  paper i n s e r t s i n h o r i z o n t a l t r a y s 1969(a)) w i t h a 10.8%  (w/v)  u t i l i z i n g Whatman  ( a f t e r Selander and  #4 Yang,  starch gel.  B u f f e r s used were s i m i l a r to those used by Selander Yang  (1969(b))  the b r i d g e  for esterase  b u f f e r was  electrophoresis.  and  Specifically,  composed of s o l u t i o n A and  the  gel  16  b u f f e r was composed o f s o l u t i o n B and A i n a 9:1 r a t i o . S o l u t i o n A was composed o f 0.03 M l i t h i u m hydroxide hydrate)  (mono-  and 0.19M b o r i c a c i d , pH 8.1; s o l u t i o n B was  0.008M monohydrate c i t r i c a c i d and 0.05M TRIS, pH 8.4. E l e c t r o p h o r e s i s was c a r r i e d out f o r 2.5 hours a t 350 volts  (and approximately  50 amps.)  Gels were kept c o o l w i t h  cracked i c e . The  g e l s were s t a i n e d with the multi-purpose p r o t e i n  s t a i n nigrosene  (c)  (Brewer, 1970).  Geographical L o c a t i o n s  ( F i g . 3)  G. a c u l e a t u s of the t r a c h u r u s form were c o l l e c t e d , i n salt-water  (Burrard I n l e t ) north o f the F r a s e r R i v e r ' s  plume, m i g r a t i n g i n t o freshwater  to breed  (Little  Campbell  River) south o f the F r a s e r R i v e r , and from an unusual p e r jnenant freshwater  situation  (Lake Erroch) .  The Lake E r r o c h f i s h are unusual resemble  completely  ( p h e n o t y p i c a l l y ) the anadromous t r a c h u r u s form o f  the f i s h and, y e t are found likely  because they  i n freshwater.  I t i s very  t h a t t h i s p o p u l a t i o n i s a marine r e l i c t s i m i l a r to  o t h e r s found.in the area, such as the r e l i c t p o p u l a t i o n o f long f i n n e d smelt, S p i r i n c h u s t h a l e i c h t h y s , found i n nearby H a r r i s o n Lake,  (Dryfoos, 1965).  T h i s p o p u l a t i o n c o u l d be  used t o p r o v i d e a check on any allozyme  c h a r a c t e r i s t i c of  e i t h e r l e i u r u s freshwater p o p u l a t i o n s o r t r a c h u r u s anadromous p o p u l a t i o n s , p a r t i c u l a r l y i f there was some evidence the polymorphism was r e l a t e d to environmental  that  adaptation.  FIGURE 3 Geographic L o c a t i o n s o f the S t i c k l e b a c k Sampled f o r the Hemoglobin Polymorphism  Populations  T h i s map r e p r e s e n t s the south Vancouver I s l a n d , lower F r a s e r R i v e r r e g i o n o f B r i t i s h Columbia. S c a l e : 1 cm equals 9 km. Sample l o c a t i o n s : 1. B u r r a r d I n l e t ; 2. Marion Lake; 3. Mike Lake; 4. Lake E r r o c h ; 5. Pemberton D i t c h ; 6. L i t t l e Campbell R i v e r ( l e i u r u s ) ; 7. L i t t l e Campbell R i v e r ( t r a c h u r u s ) ; and 8. C r y s t a l Lake.  Freshwater  a c u l e a t u s o f the l e i u r u s form were c o l -  l e c t e d from Marion Lake and Mike Lake of  the F r a s e r ) ; Pemberton D i t c h  F r a s e r ) ; and C r y s t a l Lake  (Lower Mainland north  (Lower Mainland south of the  (Vancouver I s l a n d ) .  I t should be  noted t h a t the Marion Lake f i s h were o r i g i n a l l y a Vancouver I s l a n d p o p u l a t i o n from Chemainus  stocked w i t h  Lake.  20  3.  Results  The  v i s u a l r e s u l t s of e l e c t r o p h o r e s i s corresponded to  the r e s u l t s achieved  by s t a i n i n g the g e l s with  nigrosene.  That i s , because hemoglobin i s c h a r a c t e r i s t i c a l l y red; c o u l d see the banding p a t t e r n s without simply  staining.  Staining  improved the r e s o l u t i o n of the bands. F u r t h e r ,  agreement of p a t t e r n s a t t e s t e d to the p u r i t y and  one  the  composition  of the hemoglobin. The No  samples are summarized i n Table apparent v a r i a t i o n was  b a s i s of age except  one  detected  1. i n any  g e l s on  ( l e n g t h ) , sex, h a b i t a t or season.  the  A l l gels  ( f i g . 4 ) i l l u s t r a t e d the four bands c h a r a c t e r i s t i c  of I t a l i a n phenotype A.  T h i s aberrant g e l was  a sample of lake E r r o c h f i s h . hundred o t h e r f i s h examined  Considering  produced from  that several  (trapped a t the same time) d i d  not e x h i b i t any v a r i a t i o n and  the e i g h t v a r i a b l e i n d i v i d u a l s  were a l l formed on the same g e l , i t seems u n l i k e l y t h a t the aberrant g e l r e p r e s e n t e d  r e a l genetic information.  w i l l be s a i d of t h i s i n chapter  four.  F u r t h e r experiments were performed v a r y i n g the time of a s i n g l e sample of blood. generated  w i t h i n a reasonable  samples were l e f t  More  handling  No v a r i a t i o n c o u l d  amount of time  be  (one hour). I f  f o r longer p e r i o d s of time p o l y m e r i z a t i o n  d i d occur but with no c o n s i s t e n t p a t t e r n . than f o u r components d e t e c t e d .  Never were fewer  Another p o s s i b i l i t y  that I  21  TABLE I . Summary o f Hemoglobin Sample L o c a t i o n s and R e s u l t s  POPULATION  SALTWATER OR FRESHWATER  FORM  HABITAT  SAMPLE SIZE  DATE  BLOOD TYPE  Burrard Inlet  saltwater  trachurus coastal  32  Jan.1975  type A  Marion Lake  freshwater  leiurus  lake  90  July,1975  type A  Mike Lake  freshwater  leiurus  lake  13  Nov.1975  type A  saltwater relict i n freshwater  trachurus lake  300  Jan.,May Oct.1975 June,1976  type A  Pemberton Ditch  freshwater  leiurus  stream  12  May,1976  type A  Little Campbell R.  freshwater  leiurus  stream  30  spring1975  type A  Little Campbell R.  saltwater  trachurus coastal  105  springl975  type A  Crystal Lake  freshwater  leiurus  37  summer1975  type A  Lake Errock  lake  22  FIGURE  The Aberrant  A  Gel  T h i s f i g u r e i s a d i a g r a m a t i c r e p r e s e n t a t i o n of the a b e r r a n t g e l taken from a p o p u l a t i o n o f Lake E r r o c h f i s h . A l l bands were a n o d a l , S i s the s t a r t i n g p o i n t , pH 8.4. Four i n d i v i d u a l s were c h a r a c t e r i z e d by Hb with 2 components, f o u r i n d i v i d u a l s had t h r e e components and seven i n d i v i d u a l s had f o u r components(the t y p i c a l type A p a t t e r n ) .  23  I I I  II  I  llll I i  III  I  III III III  I I I  III  I  II mi  i i cn  III  III  llll I mi i in i +  c o n s i d e r e d was the degree using this  that  t h e f o u r e l e c t r o p h o r e t i c bands  of oxygenation  o f t h e Hb  c y a n i d e as a c o m p l e x i n g n o t t o be  the  case.  agent  molecule. (Sharp,  reflected  Experiments  1973)  showed  .25  4.  D i s c u s s i o n and C o n c l u s i o n s  In one e l e c t r o p h o r e t i c g e l (15 f i s h ) of Lake E r r o c h t r a c h u r u s 8 i n d i v i d u a l s appeared t o be a b e r r a n t . banding  p a t t e r n e x h i b i t e d by these aberrant samples was  s i m i l a r t o the I t a l i a n r e s u l t s  (at pH 3.5).  Three t h i n g s are unfortunate about t h i s Firstly,  The  occurrence.  the f i s h are k i l l e d to remove the blood.  Secondly,  my own o b s e r v a t i o n s i n d i c a t e d t h a t samples c o u l d not be stored.  T h i r d l y , t h i s i n c i d e n t o c c u r r e d e a r l y i n the  experimentation unusual.  and d i d not appear  The sum o f those three f a c t s , means t h a t there are  no second chances a t determining specific  (at the time) t o be  the banding  pattern of a  individual.  Another r e s e a r c h e r  (Sharp, 1973) has r e p o r t e d t r a n s i e n t  s i t u a t i o n s o c c u r r i n g due to the innate i n s t a b i l i t y o f f i s h blood.  Sharp  (1973) recommends e i t h e r c a r r y i n g out e l e c t r o -  p h o r e s i s immediately  upon e x t r a c t i o n of the blood from the  l i v e animals o r g e n e r a t i n g met-hemoglobin by complexing the Hb molecule  w i t h cyanide.  By c a r r y i n g out both these  alter-  n a t i v e s I found t h a t s t i c k l e b a c k hemoglobin e x h i b i t s the same r e s u l t s when e i t h e r o f these a l t e r n a t i v e s are used. C o n s i d e r i n g t h a t o n l y a few samples  (eight) on one  s i n g l e g e l (15 i n d i v i d u a l s ) showed v a r i a t i o n when over 200 i n d i v i d u a l samples from the same p o p u l a t i o n  immediately  b e f o r e and a f t e r the aberrant g e l were monomorphic I concluded t h a t some t e c h n i c a l e r r o r had been made on t h a t  25  p a r t i c u l a r day. Obviously trachurus, exhibit  then,  C o l u m b i a where  salt-water  trachurus  and f r e s h w a t e r  leiurus a l l  freshwater  o n l y phenotype  g e n e r a l European to  in British  A  ( F i g . 2 ) , one must r e j e c t t h e  hypothesis  an anadromous e x i s t e n c e ) .  (that  the polymorphism  i s related  27  CHAPTER I I I .  1.  STICKLEBACK MUSCLE MYOGEN  Introduction Hagen  (1967) examined i n t e n s i v e l y , m o r p h o l o g i c a l ,  e c o l o g i c a l and e l e c t r o p h o r e t i c d i f f e r e n c e s between a r e s i d e n t i a l population  of l e i u r u s and an anadromous  of t r a c h u r u s i n the L i t t l e Campbell River B r i t i s h Columbia. Campbell River isolated.  population  i n Southwestern  Hagen i l l u s t r a t e d t h a t , i n the L i t t l e  the two forms of s t i c k l e b a c k are e c o l o g i c a l l y  Transfer  experiments were performed and showed  t h a t each form seemed to be w e l l adapted t o i t s own and p o o r l y adapted t o the h a b i t a t o f the o t h e r .  habitat  Hagen  concluded t h a t the two forms i n t h i s p a r t i c u l a r h a b i t a t situation,  f i t Mayrs' (1963) c r i t e r i o n as i n d i v i d u a l s p e c i e s .  Mayr (197 0) agreed with Hagen's a n a l y s i s . One of the c h a r a c t e r i s t i c s Hagen u t i l i z e d was the muscle myogen e l e c t r o p h o r e t i c p a t t e r n  ( F i g . 5 ) . He d i s -  covered t h a t , as.with.body p l a t e s on f i s h e s i n the L i t t l e Campbell River;  electrophoretic patterns  d i s t i n g u i s h each phase. an absolute  could be used t o  In f a c t , Hagen f e l t t h a t there  was  a s s o c i a t i o n between form and e l e c t r o p h o r e t i c  pattern. Jones  (unpublished) f o l l o w e d  f i s h from Paxton Lake. appear to c o - e x i s t . isolated.  t h i s work up by examining  He found t h a t i n t h i s lake two forms  He f e l t however, t h a t they were e c o l o g i c a l l y  Larson. (1976) supports t h i s view of the Paxton  Lake s t i c k l e b a c k .  Larson i l l u s t r a t e s t h a t two e c o l o g i c a l l y  28  FIGURE 5 The Muscle Myogen P a t t e r n This f i g u r e i s a diagramatic r e p r e s e n t a t i o n of the allozymic patterns of muscle myogen p r o t e i n s . The polymorphic enzyme has been i d e n t i f i e d as c r e a t i n e k i n a s e . S i s the s t a r t i n g p o i n t a t pH 8.4. Type S i s the slow a l l e l e , SF i s t h e heterozygous a l l e l e and F i s the f a s t a l l e l e .  29  pH 8.4  SF  F  30  different  forms o f s t i c k l e b a c k i n h a b i t the l a k e .  i s b e n t h i c , one l i m n e t i c .  One form  F u r t h e r , i t appears t h a t t h i s  e c o l o g i c a l s e p a r a t i o n i s based upon i n h e r e n t b e h a v i o r a l characteristics. Jones u t i l i z e d h y b r i d index  scores with muscle myogen  type as one of the c h a r a c t e r s , but Jones found precise distinction exist.  t h a t the  t h a t Hagen was able t o make d i d not  (Assuming one equates the l i m n e t i c and b e n t h i c forms  d e s c r i b e d by Larson  (.1976) with the t r a c h u r u s and l e i u r u s  forms d e s c r i b e d by Hagen ( 1 9 6 7 ) ) . evidence  t o support  stead Ives found  There i s m o r p h o l o g i c a l  t h i s assumption,  (Larson, 1 9 7 6 ) .  In-  t h a t the b e n t h i c phase had a p r o p e n s i t y f o r  the F phenotype, whereas the l i m n e t i c type had a p r o p e n s i t y f o r the S phenotype  ( F i g . 5) .  A c o m p l i c a t i o n emerged when gene f r e q u e n c i e s of the two p o p u l a t i o n s were pooled. found  The pooled gene f r e q u e n c i e s were  t o c o i n c i d e with Hardy-Weinburg f r e q u e n c i e s . How two  m o r p h o l o g i c a l l y and b e h a v i o r a l l y d i s t i n c t p o p u l a t i o n s can have a common gene with a l l e l e s a t a Hardy-Weinburg  frequency  has not been s a t i s f a c t o r i l y e x p l a i n e d . Two important  b i t s o f i n f o r m a t i o n should a l s o be con-  s i d e r e d with regard t o the work i n Paxton Lake.  Firstly,  Paxton Lake was dammed i n 1956,  effectively eliminating  c u t t h r o a t t r o u t , Salmo c l a r k i i ,  the only s i g n i f i c a n t  of s t i c k l e b a c k i n the l a k e .  Secondly,  predator  Hay and McPhail  (1975) r e p o r t e d t h a t s t i c k l e b a c k s p r e f e r to mate with  like  forms. A c c o r d i n g l y , with the c o m p l i c a t i o n s Paxton Lake system, i t was  i n v o l v e d i n the  p l a u s i b l e t h a t Hagen s 1  d e s c r i p t i o n of e l e c t r o p h o r e t i c i n f o r m a t i o n was curate  ( i . e . t r a c h u r u s e x c l u s i v e l y one  the other)  and  initial  indeed  ac-  phenotype, l e i u r u s  t h a t the Paxton Lake p o p u l a t i o n was  an aber-  r a n t p o p u l a t i o n where premating i s o l a t i n g mechanisms had broken down between anadromous f i s h trapped  by the dam  and  the r e s i d e n t p o p u l a t i o n . I. .therefore..decided-to., t e s t Hagen s i n i t i a l 1  that the c h a r a c t e r i s t i c phenotype F  assumption  ( F i g . 5) t y p e f i e d f r e s h  water l e i u r u s whereas the phenotype S t y p e f i e d anadromous trachurus.  F u r t h e r , as Jones' work i m p l i e d , there i s o f t e n  a d i f f e r e n c e between l i f e s t y l e s of the two  extreme phases.  Higher p l a t e d animals tend to be l i m n e t i c p l a n k t i v o r e s whereas lower p l a t e d animals tend to be b e n t h i c  browsers.  A c c o r d i n g l y , to i n c o r p o r a t e t h i s aspect  i n t o the  a range of p o p u l a t i o n s  (mean) p l a t e d  was  ( i n ..other words, to t e s t i f the polymorphism  was  examined  from low  to high  hypothesis  r e l a t e d to l i f e s t y l e I assumed t h a t i n c r e a s e d p l a t e number r e f l e c t e d an i n c r e a s e d p e l a g i c n a t u r e ) .  F i s h were a l s o  examined from a v a r i e t y of gross h a b i t a t types. It  (Table  2)  should be noted t h a t Jones i d e n t i f i e d the p o l y -  morphic components as being the enzyme c r e a t i n e k i n a s e , other r e s e a r c h e r s  ( G o s s e l i n - Rey,  e t al., 1968)  have  as  32  done w i t h specific  other  fishes.  T h i s was  performed using  enzyme  stains.  Creatine kinase regeneration  i s one o f t h e enzymes w h i c h a i d s i n t h e  o f ATP d u r i n g m u s c u l a r a c t i v i t y  (Bergmeyer,  1974) . c r e a t i n e p h o s p h a t e + ADP  >creatine  + ATP  33  TABLE I I . A Summary o f Muscle Myogen P o p u l a t i o n L o c a t i o n s , Forms and H a b i t a t s  POPULATION  FORM  SALT/FRESHWATEE HABITAT  POTENTIAL PREDATORS  trachurus  saltwater  coastal  present  Mike Lake  leiurus  freshwater  bog l a k e  present  Lake E r r o c h  trachurus  freshwater  lake  present  Pemberton D i t c h  leiurus  freshwater  stream  present  L i t t l e Campbell R. l e i u r u s  freshwater  stream  present  saltwater  coastal  present  small lake  present  Burrard  Little  Inlet  Campbell R. t r a c h u r u s  Dougan Lake  leiurus  freshwater  Chemainus Lake  leiurus  freshuatpr  Mesachie Lake  semiarmatus  freshwater  Beck Lake  leiurus  freshwater  D i v e r Lake  leiurus  freshwater  Brannen Lake  leiurus  freshwater  p i - n h a h l <=  large lake lake small lake lake  present present present present  2.  Materials  (a)  Field  The and  Methods  Techniques  Fishes described  and  were c o l l e c t e d u t i l i z i n g  i n Chapter fish  frozen  methods  previously-  II.  were e i t h e r k i l l e d  on  the  dry  i c e or  frozen  samples were t h e n  stored  until  i m m e d i a t e l y upon a f t e r blood  capture  extraction.  electrophoresis  was  The  carried  out.  (b)  Electrophoretic At  block  the  appropriate  o f m u s c l e was  each f i s h . The  Techniques  taken that only m i x e d w i t h an  p h o s p h a t e b u f f e r a t pH 10 for  seconds.  The  7.5  and  left  caudal  m u s c l e was  centrifuged  soaked  i n the  g e l s were s u b j e c t e d s t a i n e d w i t h the  300  hours.  Paper  to e l e c t r o II.  The  Gels  were  s t a i n nigrosene.  Location  were c o l l e c t e d f r o m  western B r i t i s h  0°C.  i n chapter  v o l t s f o r two  a l l purpose p r o t e i n  Geographical Fishes  to  described  0.055M  5000 r.p.m.  s u p e r n a t a n t were s u b j e c t e d  p h o r e s i s i n s t a r c h g e l , as  of  approximately  at at  a  removed.  homogenized f o r  homogenate was  region  e q u a l volume o f  5 minutes i n a r e f r i g e r a t e d c e n t r i f u g e  inserts  (c)  s a m p l e s were thawed and  removed f r o m t h e  C a r e was  m u s c l e was  time the  Columbia.  (Fig.  several 6)  locations in  south-  35  Stickleback  u s e d were, o f c o u r s e ,  hemoglobin e l e c t r o p h o r e s i s . reflecting  a cline  from s o u t h e a s t e r n reflected  (d)  f i s h e s from  Vancouver  Island  (Table  3).  a c r o s s - s e c t i o n o f f o r m s and h a b i t a t  Sample  populations  o f mean p l a t e numbers, were c o l l e c t e d The samples types.  Storage  Samples were s t o r e d  a t -30°C.  performed t o t e s t anecdotal only  Also,  those c o l l e c t e d f o r  be s t o r e d  briefly.  A small  information  that  A sample o f t w e l v e  e x p e r i m e n t was samples f i s h was  i n e a r l y O c t o b e r , 1975, thawed f o r e l e c t r o p h o r e s i s later,  refrozen,  could frozen  one week  and t h e n r e - e x a m i n e d e a r l y September, 1976.  36  FIGURE 6 Geographical Locations of S t i c k l e b a c k Populations Sampled f o r the Muscle Myogen Polymorphism S c a l e : 1 cm equals 9 km. Sample l o c a t i o n s : 1. B u r r a r d I n l e t ; 2. Mike Lake; 3. Lake E r r o c h ; 4.Pemberton D i t c h ; 5. L i t t l e Campbell R i v e r l e i u r u s ; 6. L i t t l e Campbell R i v e r t r a c h u r u s ; 7. Dougan Lake; 8. Chemainus Lake; 9. Mesachie Lake; 10. Beck Lake; 11. D i v e r Lake; 12. Brannen Lake; 13. Paxton Lake ( s t u d i e d by Jones and L a r s o n ) .  37  TABLE I I I . A Summary o f Vancouver I s l a n d P o p u l a t i o n s  Sampled  By assuming t h a t i n c r e a s i n g p l a t e number per i n d i v i d u a l r e f l e c t s i n c r e a s i n g p e l a g i c n a t u r e one can t e s t the h y p o t h e s i s t h a t muscle myogen genotypes a r e r e l a t e d t o l i f e s t y l e . Mean p l a t e numbers per i n d i v i d u a l were p r e v i o u s l y determined by J.D. M c P h a i l (pers.com.).  LOCATION Chemainus  Lake  Dougan Lake  MEAN PLATE NO. 6.0 7.3  Beck Lake  10.0  Brannen Lake  11.2  D i v e r Lake  11.8  Mesachie Lake  14.5  PER INDIVIDUAL  39  3.  Results Banding p a t t e r n s were s i m i l a r to those observed  Jones  (unpublished)  and Hagen (1967),  ( F i g . 5).  by-  Variation  o c c u r r e d a t a s i n g l e l o c u s with three banding p a t t e r n s possible;  SS,  SF and  FF.  F i v e of the twelve p o p u l a t i o n s examined were p o l y morphic. In each of these cases the gene f r e q u e n c i e s d i d not d i f f e r s i g n i f i c a n t l y from the Hardy-Weinburg f r e q u e n c i e s , (Table 4) . . The  f i v e polymorphic p o p u l a t i o n s were not  a c t e r i z e d by any gross h a b i t a t type, p o p u l a t i o n s , two  lake p o p u l a t i o n s , and one  an anadromous p o p u l a t i o n were V a r i a t i o n was season.  (Table 4).  char-  Two  creek  i n d i v i d u a l from  polymorphic.  not a p p a r e n t l y based upon age  ( s i z e ) , or  ( F i g . 7 (a) (b) ) '.'  With regard to storage, experiments were performed r e examining r e f r o z e n samples a f t e r a year o f s t o r a g e . R e s u l t s a f t e r one year were, a c t u a l l y ,superior to the r e s u l t s , i n i t i a l l y obtained.  In other words, i n i t i a l l y  ten  i n a sample of twelve were c l e a r l y readable; one twelve  individuals year l a t e r  samples were c l e a r l y readable with the l a t t e r  situ-  a t i o n r e i t e r a t i n g the. i n t e r p r e t a t i o n of the former ten results.  40 TABLE IV. Muscle Myogen Frequencies Theoretical a l l e l e frequencies were calculated from the r e a l data then expected Hardy-Weinberg frequencies were extrapolated. The r e s u l t i n g t h e o r e t i c a l values were compared with the r e a l frequencies using the Chi test. S t a t i s t i c a l l y there was no discrepancy between the r e a l and theoretical values(figures i n parentheses r e f l e c t expected values).  POPULATION  SAMPLE SIZE  CHI AND PROBABILITY 2  S (SLOW)  S/F  F (FAST)  Burrard Inlet  32  32  -  -  Lake Erroch  45  45  -  -  L i t t l e Campbell R. (leiurus)  45  8(8.91)  24(22.23)  13(13.86)  L i t t l e Campbell R. (trachurus)1975  34  33  1  -  L i t t l e Campbell R. (trachurus) 1976  45  45  -  -  Mike Lake  13  13  -  -  Pemberton Ditch  12  5(3.96)  -(0.48)  Chemainus Lake  30  30  -  -  Dougan Lake  28  12(12.88)  14(12.04)  2(2.80)  0.61, P = .45  Beck Lake  28  8(8.12)  14(14.00)  6(5.88)  0.004,P = .95  Diver Lake  30  30  -  -  Brannen Lake  30  30  -  -  Mesachie Lake  45  45  -  -  7(7.44)  0.28,P = .65  0.78, P = .40  41  FIGURE 7 Age  and  Season  F i g u r e 7a.: C o n s i d e r i n g l e n g t h to be synonymous w i t h age within a g i v e n p o p u l a t i o n ( e g . Beck Lake) a s t a t i s t i c a l t e s t was performed t o see i f t h e r e was any i n d i c a t i o n o f a r e l a t i o n s h i p between the muscle myogen polymorphisms and o n t o g e n e t i c development. No r e l a t i o n s h i p was found t h e r e f o r e i t was concluded t h a t the p r e s e n c e of the various p r o t e i n types was independent of age. F i g u r e 7b.: Polymorphic g e l s were d i s c o v e r e d at v a r i o u s times of y e a r . T h i s would seem to imply that the polymorphisms were not seasonally induced.  42  (A)  ANALYSIS OF LENGTH (AGE) VERSUS GENOTYPE GENOTYPE F 30.8 24.1 25.1 51.4 29.3 28.3  GENOTYPE SF 30.3 27.3 27.7 24.4 24.2 35.0 54.3 35.8 34.7 36.1 37.1 38.4 34.2 30.7  GENOTYPE S ( l e n g t h i n mm.) 28.6 28.4 27.5 23.4 23.4 37.3 35.6 31.1 235.3 x = 29.4  470.2  189.0 x = 31.5  x = 33.6  ANOVA  SUM OF SQUARES  DEGREES FREEDOM  MEAN SQUARE  BETWEEN MEANS  90.2  2  45.1  WITHIN GROUPS  1433.5  25  57.3  SOURCE  Test H : o  Test  F  l  =  h  statistic  =  H^ = n o t t h e same  2 F = Sm^ , c r i t i c a l r e g i o n Sp  F<F  >  9  9  (  2  }  2  5  )  = 5.6  The v a l u e f o r F a t Beck Lake i s 0.8 t h e r e f o r e one must a c c e p t t h e h y p o t h e s i s t h a t t h e means a r e e q u a l . (B)  Time o f y e a r sample t a k e n May 1976 J u l y 1973 August 1976 September 1976  polymorphic p o p u l a t i o n Pemberton D i t c h P a x t o n Lake L i t t l e Campbell R. l e i u r u s Beck and Dougan Lake  43  4.  D i s c u s s i o n and Obviously,  and  Conclusion  Hagen's o r i g i n a l assumption, t h a t l e i u r u s  trachurus are t y p e f i e d by s p e c i f i c e l e c t r o p h o r e t i c  banding p a t t e r n s , must be r e j e c t e d f o r the s p e c i e s complex as a whole. animals  Both the h i g h e s t p l a t e d and  lowest  plated  ( i . e . ocean trachurus vs. Chemainus Lake l e i u r u s )  are e l e c t r o p h o r e t i c a l l y s i m i l a r .  Indeed, even samples from  the l e i u r u s zone of the L i t t l e Campbell R i v e r do not comp l e t e l y r e f l e c t Hagen's o r i g i n a l  impression.  F u r t h e r , using f i s h l e n g t h w i t h i n a p o p u l a t i o n as  an  i n d i c a t i o n of f i s h age one must conclude t h a t the p o l y morphism i s not ontogenetic morphism i s present  i n nature.  i n both small and  That i s , the  poly-  large fishes.  When Jones c a r r i e d out h i s e l e c t r o p h o r e s i s , f i s h s m a l l e r than 15 mm  were completely  ( P e r r i a r d e t .al., 1972)  homogenized.  Researchers  have shown enzyme v a r i a b i l i t y between  tissues, within a single individual. (Eppenberger e t al.,. 1971;  Indeed some r e s e a r c h e r s  L y s l o v a , 1971)  have found  this  very phenomenon o c c u r r i n g with the enzyme c r e a t i n e k i n a s e . By t a k i n g muscle t i s s u e from the l e f t caudal peduncle area t h i s c o m p l i c a t i o n was  avoided.  A c c o r d i n g l y then, one  assume t h a t the v a r i a b i l i t y observed was I f one  real.  examines ( F i g . 8) the g e o g r a p h i c a l  of the p r o t e i n polymorphism one  can  distribution  notes there i s no  indication  of the polymorphism r e f l e c t i n g ranges of i n d i v i d u a l demes of  FIGURE  8  G e o g r a p h i c a l L o c a t i o n s o f the Polymorphic  Populations  Populations 4, 5, 7, 10, and 13 (as w e l l as one i n d i v i d u a l from the sample from p o p u l a t i o n 6) were d i s c o v e r e d t o be polymorphic. The s i t e s o f these p o p u l a t i o n s a r e c i r c l e d . N o geographic t r e n d i s apparent.  .45  Gasterosteus  aculeatus.  rough estimate differ  of  from the  Also, considering altitude  i n v a s i o n time,  older populations  assumed p a r e n t a l p o p u l a t i o n s .  as do  a not  (ocean  trachurus) A comparison of gross stream, the  lake,  salt-water,  d i s t r i b u t i o n of  correlation.  habitat characteristics freshwater,  etc.) with  the polymorphism p r o v i d e s  no  (i.e.  regard obvious  to  47  CHAPTER IV.  My  GENERAL DISCUSSION  study contains r e s u l t s  different  levels.  T h i s c a n be  that  are important a t  summarized  i n the  several  following  way. (a)  'Stickleback  Hemoglobin  Southwestern B r i t i s h  Columbia G a s t e r o s t e u s a c u l e a t u s  have monomorphic h e m o g l o b i n . s e r v e d , on a s i n g l e  occassion,  E u r o p e a n work, p a r t i c u l a r l y arities  The  results  some d o u b t on  the  when one  c o n s i d e r s the  simil-  pattern  ( R a u n i c h e t al«, 1972)  t h e i r work s h o u l d be r e - e x a m i n e d results. ation  However, i t w o u l d  o f t h e Hb p o l y m o r p h i s m in a single  8.4  a t pH  and  3.5.  to double check  simply  p e r f o r m e d b r e e d i n g e x p e r i m e n t s and  with v a r i a t i o n  a t pH  appear u n l i k e l y  i s a technique a r t i f a c t ,  ratios  polymorphism  casts  between t h e t r a n s i e n t  European  transient  that  Obviously their  their  situ-  because  the  Europeans  found t h a t  the  offspring  corresponded to a  gene.  the  situation  I t s h o u l d be n o t e d  o n l y o f f s p r i n g were e x a m i n e d .  It i s clear,  aspect of stickleback  i s t o be p e r s u e d i t s h o u l d  done i n t h e A d r i a t i c  genetics  that  that  a r e a , p e r h a p s on a more  i f this be  physiological  level. As fit  I mentioned  traditional  fore,  that  distributed  i n Chapter I, s t i c k l e b a c k  s p e c i e s paradigms.  comparative genetic p o p u l a t i o n s has  I t would  do n o t seem t o appear,  there-  i n f o r m a t i o n among w i d e l y  interest.  48  Some d i s c u s s i o n use  ( A v i s e , 1975) h a s o c c u r r e d  of electrophoresis i n systematics,  several different compared and  loci  species a t a single  Sharp  species.  g l o b i n molecules Coast,  different  kinase three are  when  Other r e s e a r c h e r s  locus.  Tsyuki  have  e t a l . , (1963)  The f o r m e r g r o u p e x a m i n e d  and f o u n d  t h a t t h e b a n d i n g p a t t e r n s were  f o r each s p e c i e s , both Ferguson  patterns  t o suggest  known t o be e n v i r o n m e n t a l l y precedents  c o u l d be j u s t i f i e d banding p a t t e r s all  freshwater  and  a l l southern  reinforcing  electro-  (and some s a l t w a t e r )  evidence  These  coregonid  w i t h many o f t h e induced.  the s i m i l a r i t y  bands, t h r e e  British  status f o r  i n mind, i t a p p e a r s t h a t one  i n saying,  (anodal  species  fishes.  variable morphologically,  With these  several  (general p r o t e i n s , esterases, c r e a t i n e  f o r m s of. a complex o f c o r e g o n i d  variations,  very  i n t e r m s o f component  (1974) c o n s i d e r e d  and p h o s p h o g l u c o m u t a s e )  extremely  hemo-  o f seven salmonids o f the n o r t h e a s t e r n  number and s h a p e . phoretic  particularly  (1973) f o r example have compared h e m o g l o b i n s among  various related  Pacific  a r e examined.  supporting the  Columbia  among  hemoglobin  l a r g e , one s m a l l ) o f European s t i c k l e b a c k s  sticklebacks provides  that the populations  i n question  belong  t o t h e same s p e c i e s , (b)  S t i c k l e b a c k M u s c l e Myogen Two p o s s i b i l i t i e s  distribution populations.  remain with  of the c r e a t i v e kinase  respect alleles  to explaining the i n stickleback  49  Firstly, adaptation  i t i s possible  to a s u b t l e  that  the  environmental character.  hypothesis i s extremely d i f f i c u l t impossible  to  to  possibility  morphism i s s e l e c t i v e l y support t h i s point  neutral.  of view.  the  however, w i t h  alleles  Further,  are  Redfield et  result  was  over a period locations  of  proof  time, to  of polymorphic  I t has 1976; is  fish  reported  been s t a t e d U t h e and  see  extremely rare.  One  i n the  grouse of  grouse  the  polymorphism.  situ-  This  i f gene f r e q u e n c i e s ,  Study and  change  or  this situation. i s that  stickleback  t o have a m u s c l e myogen literature  R y d e r , 1970);  a similar  i n blue  stickleback populations  i n the  all  implausible  s e l e c t i v e maintenance.  Another i n t e r e s t i n g p o i n t first  samples  i n s t i c k l e b a c k except a l l  of  remain s t a b l e , would c l a r i f y  the  the  from a wide range  Results  examined c o n t a i n e d as  to  neutral.  plasma polymorphism  Vancouver I s l a n d .  cited  seem n o t  a l . , (1972) a t t e m p t e d  a t i o n were s i m i l a r t o t h o s e populations  poly-  populations  Admittedly  i t would  (Dendragapus o b s c u r u s ) p o p u l a t i o n s on  the  f i v e polymorphic populations,  selectively  study examining a blood  habitats  probably  T h e r e i s some e v i d e n c e  A l l polymorphic  Hardy-Weinberg e q u i l i b r i u m  that  This  also exists that  were i n H a r d y - W e i n b u r g e q u i l i b r i u m .  in  t e s t , and  reflects  discount.  Secondly, the  were s m a l l ,  polymorphism  (Morgan and  t h a t m u s c l e myogen  is inclined  are  not  polymorphism. Ulanowicz, polymorphism  t o comment t h a t  muscle  50  myogen p o l y m o r p h i s m s c a n than  uncommon c a t e g o r y  be moved f r o m t h e  since at least  r a r e to the  s i x species are  t o have p o l y m o r p h i s m s o f t h i s  type.  species  the p h y l o g e n e t i c  ( T a b l e 5)  illustrates  r a n g e encompassed by  not.been p o s i t i v e l y  all  these  identified  exhibiting  fishes,  fishes,  and  piscivores  among t h e s e  river  a reflection  lines.  fishes,  of the  The  species  structural  Creatine kinase  t o have a d i m e r i c  and  with  d i m e r i c the  that this  limitations  however t h i s m u l t i t u d e  of the  implying non-dimeric  out  t h i s monomeric n a t u r e  the  phylo-  well studied  I t has  of  a l w a y s been  the  s t i c k l e b a c k polymorphism.  heterozygous  dimeric  form  structure could  technique  structure.  (S/F)  (Fig. 9).  on  one  May  would It is be  occassion,  o f e x a m p l e s p r o v i d e s many  all  this  t h e Duchenne t y p e  t h r e e bands i n s t e a d o f two  plausible  Onchorynchus.  factor  s t r u c t u r e (Kuby e t a l . , 1 9 6 2 ) .  compatible  enzyme was  have c o n t a i n e d  o n l y common  is a relatively  (Bergmeyer, 1 9 7 4 ) .  T h i s i d e a i s not  p e l a g i c ocean  information across  muscular dystrophy  masked by  in  sampled.  its. a s s o c i a t i o n with  remotely  polymorphism  are a l l t e l e o s t s ,  enzyme b e c a u s e o f  the  environmental  k i n a s e polymorphism a l s o emphasizes  lack of biochemical  If  and  these  as c r e a t i n e k i n a s e  herbi vores.  s p e c i e s i s t h a t they  The.creatine  thought  of  the polymorphism i n c l u d e s a l t w a t e r  fishes,, freshwater  genetic  (The  known  examples).  Species  c o u l d be  A comparision  t h i s polymorphism.  has  less  repetitions  (1975) a l s o p o i n t e d  of c r e a t i n e kinase  i n the  genus  Many e l e c t r o p h o r e t i c s t u d i e s have p o i n t e d  out  TABLE V. A Comparison of the S i x Species Demonstrating a Similar Muscle Myogen Protein Polymorphism  SPECIES  LOCATION  HABITAT  ECOTYPE  copepods shrimp mysids  Menidia menidia  A t l a n t i c coast  estuarine areas  Catostomus catostomus  North America and S i b e r i a  freshwater  bottom browser  Anoplopoma fimbria  North P a c i f i c  saltwater  pelagic  Sebastodes elongatus  North America P a c i f i c coast  saltwater  50-200 fathoms  Stizostedion vitreum  c e n t r a l and east North America  freshwater  pelagic schooler  piscivore  freshwater and anadromous  benthic/pelagic  ostracods larvae plankton  Gasterosteus aculeatus  northern hemisphere  schooling  FOOD  REFERENCES Morgan and Ulanowicz, 1976. Liem and Scott,1966  McPhail and Lindsey, 1970. amphipods insect larvae Tsuyuki et al.,1967.  piscivore  -  Tsuyuki and Roberts, 1969. Hart,1973.  Tsuyuki et a l . , 1969. Hart,1973. Uthe and Ryder, 1970. McPhail and Lindsey,1970.  McPhail and Lindsey, 1970.  52  FIGURE  9  H y p o t h e t i c a l P a t t e r n f o r D i m e r i c Quarternary of the Enzyme C r e a t i n e Kinase  Structure  S r e p r e s e n t s the s t a r t i n g p o i n t . Homozygous S i s r e p r e s e n t e d by component a, homozygous F i s r e p r e s e n t e d by component c and heterozygous SF has both these components p r e s e n t p l u s component b (which would be a composite form of a and c ) .  t  S  SF  F  54  s i m i l a r molecular  i n c o n g r u i t i e s but  have i n v e s t i g a t e d f u r t h e r .  For  few  researchers  example c o m p a r i s o n s  mammalian h e m o g l o b i n seems t o have c a u s e d  people  the p h y l o g e n e t i c c o n s i s t e n c y o f the q u a r t e n a r y all  with  regard  the  taxonomic arguments c i t e d  to hemoglobin.  to the d i s t r i b u t i o n  That  to support  members o f t h e (c)  and  similar  remote from the ocean.  s t r u c t u r e of  in this  same argument c a n The  t r a c h u r u s , were  to the  chapter  be  applied  extreme  forms  electrophoretically were  leiurus populations  T h i s would a l s o p r o v i d e  the view t h a t l e i u r u s  and  most  reinforcing  trachurus  are  same s p e c i e s ,  Future,Research Consider  stated  the b l o o d  earlier,  checked.  The  of  i n Europe.  I  following bits  should  check i s p o s i t i v e ,  of  research.  would  involve  c h a r a c t e r i z i n g each e l e c t r o p h o r e t i c hemoglobin in.Gasterosteus  (Fig. 2). or  the  As  the polymorphism  o p e r a t i o n I would p e r f o r m  component f o u n d  hypothesis  authenticity  perform  first  biochemically  B  the  polymorphism  A s s u m i n g t h e outcome o f t h i s  I would then  and  of  to generalize  i s , s a l i n e p o p u l a t i o n s of trachurus  electrophoretically  evidence  The  of c r e a t i n e kinase.  stickleback, leiurus  similar.  be  to  p r o t e i n s of a l l animals. Consider  of  seem  a c u l e a t u s p h e n o t y p e s A,  T h i s would e i t h e r  suggest  some o t h e r  support  alternative  Further,  I would i n c l u d e a d e t e r m i n a t i o n  weights  of each of the  standing of t h e i r  the  European  hypothesis.  of the  molecular  components so t h a t a c l e a r e r  structural  nature  AB  m i g h t emerge.  under(Perhaps  55  even going as f a r as sequencing the p r o t e i n ) . Then, armed w i t h a new h y p o t h e s i s or r e i n f o r c e d o l d h y p o t h e s i s I would perform t r a n s f e r experiments.  I would  t r a n s p o r t o f f s p r i n g of known parentage i n t o s a l t ,  brackish  and  freshwater environments t o determine whether o r not the  oxygen c a p a c i t i e s were g e n e t i c a l l y predetermined or the r e s u l t o f a c c l i m a t i z a t i o n , (which would a l s o be a g e n e t i c phenomenon).  Guernsey and Puluhowich  t h i s l a t t e r phenomenon i n blood  (1975)  illustrated  oxygen c a p a c i t i e s o f the  /American e e l ( A n g u i l l a r o s t r a t a ) i n s a l t , b r a c k i s h and freshwater environments.  A check f o r t h i s phenomenon i n  B r i t i s h Columbia s t i c k l e b a c k may a l s o prove i n t e r e s t i n g . Would anadromous and freshwater s t i c k l e b a c k s  (with the same  e l e c t r o p h o r e t i c components) have d i f f e r e n t oxygen c a p a c i t i e s based upon e l e c t r o p h o r e t i c a l l y u n d e t e c t a b l e g e n e t i c  dif-  ferences . Another p o s s i b l e p r o j e c t c o u l d i n v o l v e a  biochemical  comparison between the hemoglobin o f the t h r e e s p i n e back (G. aculeatus) pungitius) .  and the n i n e s p i n e  stickleback  diets,  (Pungitius  Lewis e t al., (1972) observed t h a t i n England  these two s p e c i e s are o f t e n found together habitats.  stickle-  i n the same  They are a l s o known to have v i r t u a l l y  (Hynes), 1950).  The q u e s t i o n  s p e c i e s avoid, competition? w i t h i n those h a b i t a t s , niche  arose,  identical  how do these  Lewis e t a l . , observed separation  the b a s i s o f oxygen a v a i l a b i l i t y .  that  appeared to occur on  Pungitius  pungitius  5; 6  appeared  t o be more t o l e r a n t  physiological where t h e s e  adaptation.  species are  where t h e y  are  found  of  Since  found  alone  low  respect to biochemical  logical)  together  ( S c o t t and  e t a l . , (1972) s t a t e d i n I t a l y electrophoretically Gasterosteus Now,  similar  the  continuing  research  to the  possibly 1970).  components.  hemoglobin of  characteristics  Raunich  non-migratory  and  molecular  T h i s m i g h t answer t h e  ( i f they  S c h o l l and  individual  h e a r t and  forms?  The  insight  into  question  vary  stomach t i s s u e s  Further, electrotissues  a r i s e s , Why? and  a l l have  more k n o w l e d g e on  not  their  among  form o f c r e a t i n e k i n a s e  Obviously,  various  q u e s t i o n whether or  (1972) have p o i n t e d o u t ,  fishes.  s h o u l d m u s c l e have one t e s t e and  temperature  weights of the  components o f c r e a t i n e k i n a s e  individual  about  creatine kinase  have some s u b t l e f u n c t i o n ) .  Eppenberger  be  I f I were  of the  optima,  components a r e n e u t r a l , o r g i v e  function,  out  (physio-  I w o u l d t r y t o l e a r n more  I w o u l d e s t a b l i s h pH  optima pressure, e f f e c t s  within  carried  P u n g i t i u s hemoglobin i s  c r e a t i n e k i n a s e polymorphism?  in this  components.  and  P.  1973), i t  work c o u l d be  (Dobzhanski,  exist  aculeatus.  t h e i.Yi v-u&io b i o c h e m i c a l  phoretic  a l s o areas  Crossman,  of  exist  t h e q u e s t i o n a r i s e s what f u r t h e r work s h o u l d  done w i t h  the  and  a d a p t a t i o n , and  character displacement  because  i n Canada, a r e a s  w o u l d a p p e a r t h a t some i n t e r e s t i n g with  oxygen l e v e l s  brain,  other the  Why  as  57  structural  and b i o c h e m i c a l  more i n s i g h t  into  this regard. brain?  this question.  l e v e l s may  Other q u e s t i o n s  provide  arise i n  i s the f u n c t i o n o f c r e a t i n e kinase  i n the  Do enzymes s u c h a s c r e a t i n e k i n a s e have m u l t i p l e  functions? of  What  characteristic  Much more i n f o r m a t i o n i s n e e d e d .  electrophoresis w i l l  questions  like  be u t i l i z e d  these,have been  The  technique  more e f f e c t i v e l y  answered.  after  Literature  Cited  A s p i n w a l l , N. 1974. 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