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Geographic variation in Novumbra hubbsi Schultz 1929 (Pisches, Umbridale) : External meristic characters,… Rosenfeld, Mark Jay 1983

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GEOGRAPHIC VARIATION IN NOVUMBRA HUBBSI SCHULTZ 1929 (PISCES:  UMBRIDAE):  EXTERNAL MERISTIC CHARACTERS,  CHROMOSOMAL STATE AND NUCLEAR DNA CONTENT by MARK JAY ROSENFELD B.Sc,  U n i v e r s i t y o f Utah, 1976  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department o f Zoology)  We a c c e p t t h i s t h e s i s as conforming t o t h e required standard  THE UNIVERSITY OF BRITISH COLUMBIA October 1983 ©Mark Jay R o s e n f e l d , 1983  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree a t the  the  University  o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e f o r reference  and  study.  I further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may department o r by h i s o r her  be  granted by  the head o f  representatives.  my  It i s  understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l not be  allowed without my  permission.  Department o f  Zoology  The U n i v e r s i t y of B r i t i s h 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  DE-6  (3/81)  14 October 1983  Columbia  written  ii  ABSTRACT  Geographic V a r i a t i o n i n Novumbra hubbsi S c h u l t z 1929 (Pisces:  Umbridae):  External  Meristic  Characters,  Chromosomal S t a t e and N u c l e a r DNA Content  V a r i a t i o n i n e x t e r n a l m e r i s t i c c h a r a c t e r s , chromosomal s t a t e and nuclear  DNA c o n t e n t was examined a c r o s s  t h e range o f a r e l i c t  Novumbra hubbsi S c h u l t z , o c c u r r i n g w i t h i n t h e general Chehalis  periglacial  The  area o f the  r e f u g e near t h e t e r m i n a l moraine o f t h e l a t e  s t o c e n e Vashon G l a c i e r i n w e s t e r n Washington, U.S.A. considerable  geographic d i s t r i b u t i o n s o f mean counts f o r l a t e r a l  rays i n males as w e l l as those f o r t h e l a t e r a l  Plei-  N^. hubbsi shows  geographic v a r i a t i o n i n several external m e r i s t i c  the l a t e r a l  fish,  characters.  bars and anal f i n  s c a l e row and s c a l e s above  s c a l e row f o r males and females combined i n d i c a t e t h a t t h e  Montesano H i l l s , f o o t h i l l s o f t h e Olympic M o u n t a i n s , a r e a b a r r i e r t o gene f l o w . stocene.  T h i s b a r r i e r may have been p r e s e n t s i n c e a t l e a s t t h e P l e i L a t e r a l bar counts a r e l o w e r and d o r s a l  i n males from p r e d a t o r - f r e e  habitats.  f i n r a y counts  These c h a r a c t e r  s i d e r e d i m p o r t a n t f o r mate a t t r a c t i o n and t e r r i t o r i a l  higher  trends a r e condefense.  Reductions  i n t h e numbers o f p e c t o r a l and p e l v i c f i n rays a r e a l s o a s s o c i a t e d predator-free  habitats.  as w e l l as l a t e r a l  with  Lower numbers o f p e c t o r a l and p e l v i c f i n rays  bars and g r e a t e r numbers o f d o r s a l f i n rays may cause  f i s h t o be more prone t o p r e d a t i o n .  Character  frequencies  may change  o v e r t i m e , as i n t e r p r e t e d from comparisons o f f i s h from t h e p r e s e n t  111  s t u d y w i t h those caught i n 1947 lateral  and  1968.  The e l e v a t e d counts f o r  row s c a l e s and s c a l e rows b e f o r e the d o r s a l f i n o r i g i n i n f i s h  from the Wishkah R i v e r d r a i n a g e may  be due t o a b i o t i c  developmental  factors. As to chromosome number and morphology, v a r i a t e i n t e r s e x u a l l y and g e o g r a p h i c a l l y . p r i s e the d i p l o i d number.  The  hubbsi appears i n -  F o r t y - e i g h t chromosomes com-  fundamental number i n chromosome comple-  ments i s 80 chromosome arms, c o n t r a s t i n g w i t h a p u b l i s h e d v a l u e o f 74 arms.  The r e v i s e d fundamental number i s based on an e x t e n s i v e s e r i e s o f  measurements on 186 e l o n g a t e chromosome s e t s . N u c l e a r DNA  c o n t e n t s a r e r e p o r t e d f o r male and  from f o u r p o p u l a t i o n s . appear p r e s e n t . i s 1.68  The  female  N_. hubbsi  No i n t e r s e x u a l o r g e o g r a p h i c a l d i f f e r e n c e s  Feulgen-DNA d e t e r m i n a t i o n f o r e r y t h r o c y t e n u c l e i  pg/nucleus± 0.02(S.E.), about 80% o f a p u b l i s h e d v a l u e .  d i f f e r e n c e between d e t e r m i n a t i o n s may  The  be due to the use o f d i f f e r e n t  c o n t r o l c e l l s o r too high a r e f e r e n c e DNA  v a l u e by e a r l i e r workers.  Since  the Feulgen-DNA d e t e r m i n a t i o n i s not v e r y d i f f e r e n t from the f l u o r o m e t r i c ones i n t h i s s t u d y , as w e l l as near a v a l u e determined t i v e , the p r e s e n t DNA The observed  e s t i m a t e f o r N_. hubbsi  i s considered accurate.  c o n s e r v a t i s m i n chromosomal s t a t e and  be a consequence o f _N. hubbsi  for a close rela-  DNA  content  p e r s i s t i n g i n the same environment f o r  s e v e r a l m i l l i o n y e a r s , p o s s i b l y s i n c e the  Oligocene.  may  iv  TABLE OF CONTENTS  ABSTRACT  i i  LIST OF TABLES  vi  LIST OF FIGURES  x  ACKNOWLEDGEMENTS  x i i  Introduction  1  M a t e r i a l s and Methods Sampling  localities  11 11  C l a s s i f i c a t i o n o f sampling l o c a l i t i e s  11  P h y s i c a l and chemical d e t e r m i n a t i o n s on t h e water  11  Methods o f c o l l e c t i o n  14  Morphomeristic  14  determinations  A n a l y s i s o f m e r i s t i c data  16  Chromosome methodology  I  8  Feulgen-DNA d e t e r m i n a t i o n s  2 0  Fluorometric-DNA  2  technique  Results  2  2 3  Habitat c l a s s i f i c a t i o n  2 3  Presence o f f i s h e s o t h e r than N. hubbsi  2  3  P h y s i c a l and chemical d e t e r m i n a t i o n s on t h e water  2  5  Meristic characteristics  2  6  Sexual dimorphism o f d o r s a l and anal f i n rays Dorsal  f i n rays i n males  Dorsal f i n rays i n females  2 7  2 7  36  V  Anal f i n rays i n males  3 6  Anal  42  f i n rays i n females  Caudal f i n rays  42  Pectoral f i n rays  42  P e l v i c f i n rays  51  S c a l e rows b e f o r e t h e d o r s a l f i n o r i g i n  51  Lateral  51  row s c a l e s  S c a l e s above t h e l a t e r a l  s c a l e row  62  S c a l e s below t h e l a t e r a l s c a l e row  62  Lateral  62  bars i n males  Chromosome c h a r a c t e r i s t i c s  73  DNA q u a n t i f i c a t i o n  87  Discussion  91  The J u n c t i o n C i t y c o l l e c t i n g s i t e  91  Meristic variation  92  Chromosomal  99  state  Heteromorphy o f chromosomes 15 and 16  100  N u c l e a r DNA  101  General d i s c u s s i o n  103  Summary and C o n c l u s i o n s  106  Literature  108  Cited  vi  LIST OF TABLES TABLE 1  2  PAGE Map c o - o r d i n a t e s and s i t e d e s c r i p t i o n s f o r N. hubbsi c o l l e c t i n g s i t e s  12  C o r r e l a t i o n s between m e r i s t i c c h a r a c t e r and s t a n d a r d l e n g t h f o r _N. hubbsi  17  values  3  Selected  4  C h i - s q u a r e t e s t s on t h e d i s t r i b u t i o n s o f d o r s a l and anal f i n r a y s t a t e s compared between sexes by l o c a t i o n f o r _N. hubbsi  28  Sexual dimorphism i n t h e mean number o f d o r s a l and anal f i n rays i n H. hubbsi evaluated with t - t e s t s  29  Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r t h e number o f d o r s a l f i n rays i n male _N. hubbsi  30  Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r t h e number o f d o r s a l f i n rays i n female _N. hubbsi  31  Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r t h e number o f anal f i n rays i n male _N. hubbsi  32  Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r t h e number o f anal f i n rays i n female _N. hubbsi  33  S t a t i s t i c a l a n a l y s e s u s i n g l o g a r i t h m i c means testing f o r interpopulational differences i n t h e number o f d o r s a l f i n rays i n male N. hubbsi.  34  S c h e f f e ' s m u l t i p l e c o n t r a s t s t e s t s on t h e mean counts o f d o r s a l and anal f i n rays f o r male N. h u b b s i .  37  A n a l y s i s o f v a r i a n c e i n t h e number o f d o r s a l f i n rays i n female N. hubbsi  38  5  6  7  8  9  10  11  12  parameters o f _N. hubbsi h a b i t a t s  24  vi i  TABLE ( c o n t ' d ) 13  14  15  16  17  18  19  20 21  22  23  PAGE  S t a t i s t i c a l analyses using l o g a r i t h m i c means t e s t i n g f o r i n t e r p o p u l a t i o n a l d i f f e r e n c e s i n t h e number o f anal f i n r a y s i n male N. h u b b s i .  40  S t a t i s t i c a l a n a l y s e s u s i n g l o g a r i t h m i c means testing f o rinterpopulational differences i n t h e number o f anal f i n rays i n female N. h u b b s i .  43  Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r t h e number o f caudal f i n rays i n N. hubbsi  45  S t a t i s t i c a l a n a l y s e s u s i n g l o g a r i t h m i c means testing f o r interpopulational differences i n t h e number o f caudal f i n rays i n N. hubbsi  46  Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r t h e number o f p e c t o r a l f i n rays i n N. hubbsi  48  S t a t i s t i c a l a n a l y s e s u s i n g l o g a r i t h m i c means testing f o r interpopulational differences i n t h e number o f p e c t o r a l f i n r a y s i n N. hubbsi  49  Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r t h e number o f p e l v i c f i n rays i n N. hubbsi  52  A n a l y s i s o f v a r i a n c e and K r u s k a l l - W a l l i s t e s t s f o r t h e number o f p e v i c f i n rays i n N. hubbsi  53  Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r t h e number o f s c a l e rows b e f o r e t h e d o r s a l f i n o r i g i n i n N. hubbsi  55  S t a t i s t i c a l a n a l y s e s u s i n g l o g a r i t h m i c means testing f o r interpopulational differences i n t h e number o f s c a l e rows b e f o r e t h e d o r s a l f i n o r i g i n i i n N. hubbsi  56  Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r t h e number o f l a t e r a l row s c a l e s i n N. hubbsi  58  i ix  TABLE (cont'd) 24  25  26  27  28  29  30  31 32 33 34  35  PAGE  S t a t i s t i c a l a n a l y s e s u s i n g l o g a r i t h m i c means testing f o r interpopulational differences i n t h e number o f l a t e r a l s c a l e s i n N. hubbsi  60  Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r t h e number o f s c a l e s above t h e l a t e r a l s c a l e row i n N. hubbsi  63  S t a t i s t i c a l a n a l y s e s u s i n g l o g a r i t h m i c means testing f o r interpopulational differences i n t h e number o f s c a l e s above t h e l a t e r a l s c a l e row i n N. hubbsi  64  Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and g e o m e t r i c menas f o r t h e number o f s c a l e s below t h e l a t e r a l s c a l e row i n N. hubbsi  66  S t a t i s t i c a l a n a l y s e s u s i n g l o g a r i t h m i c means testing f o rinterpopulational differences i n t h e number o f s c a l e s below t h e l a t e r a l s c a l e row i n N. hubbsi  67  Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r t h e number o f l a t e r a l bars i n male N. hubbsi  69  S t a t i s t i c a l a n a l y s e s u s i n g l o g a r i t h m i c means testing f o r interpopulational differences i n t h e number o f l a t e r a l bars i n male N. hubbsi  70  Summary o f N. hubbsi examined f o r chromosomal states  74  Frequency d i s t r i b u t i o n o f d i p l o i d chromosome numbers o b t a i n e d from t i s s u e s o f N. hubbsi  76  Frequency d i s t r i b u t i o n o f chromosome counts from t h e g i l l e p i t h e l i u m o f N. hubbsi  79  Chromosome morphometries o f N. h u b b s i , based on 186 k a r y o t y p e s o b t a i n e d from 32 males and 30 females from s e v e r a l p o p u l a t i o n s  80  D e t e r m i n a t i o n s o f n u c l e a r DNA c o n t e n t s f o r e r y t h r o c y t e s o f N. hubbsi  88  (cont'd) S t a t i s t i c a l a n a l y s e s u s i n g the a r c s i n square r o o t t r a n s f o r m o f r e l a t i v e Feulgen-DNA v a l u e s f o r t h e n u c l e i o f N_. hubbsi erythrocytes  X  LIST OF FIGURES FIGURE I  II  III  IV  V  VI  VII 11X IX X XI  XII XIII  PAGE Map o f t h e Olympic P e n i n s u l a and a d j a c e n t r e g i o n s showing t h e range o f N_. hubbsi Geographic v a r i a t i o n i n t h e number o f d o r s a l f i n r a y s i n male j ^ . hubbsi  35  Geographic v a r i a t i o n i n t h e number o f d o r s a l f i n r a y s i n femal e N_. hubbsi  39  Geographic v a r i a t i o n i n t h e number o f anal f i n rays i n n a l e N. hubbsi  41  Geographic v a r i a t i o n i n t h e number o f anal f i n r a y s i n female.NL hubbsi  44  Geographic v a r i a t i o n i n t h e number o f caudal f i n r a y s i n N_. hubbsi  47  Geographic v a r i a t i o n i n t h e number o f p e c t o r a l f i n rays i n _N. hubbsi  50  Geographic v a r i a t i o n i n t h e number o f p e l v i c f i n r a y s i n N_.. hubbsi  54  Geographic v a r i a t i o n i n t h e number o f s c a l e rows b e f o r e t h e d o r s a l f i n o r i g i n i n N. hubbsi  57  Geographic v a r i a t i o n i n t h e number o f l a t e r a l row s c a l e s i n H. hubbsi  61  Geographic v a r i a t i o n i n t h e number o f s c a l e s above the l a t e r a l s c a l e row i n J\L hubbsi  65  Geographic v a r i a t i o n i n t h e number o f s c a l e s below t h e l a t e r a l s c a l e row i n H_. hubbsi  68  Geographic lateral  XIV XV XVI  2  v a r i a t i o n i n t h e number o f  bars i n male 'N_. hubbsi  71  Male N_. hubbsi from f o u r d i f f e r e n t r e g i o n s  72  Metaphase chromosome spreads from N_. hubbsi  77  S o r t e d k a r y o t y p e s o f N. hubbsi  82  xi  FIGURE (Cont'd) XVII  XIIX  XIX  PAGE  Idiogram o f m i t o t i c metaphase chromosomes from h u b b s i , based on t h e measurements o f arm r a t i o and r e l a t i v e l e n g t h  84  Metaphase chromosome spreads o b t a i n e d from the g i l l e p i t h e l i u m o f N_. hubbsi c a p t u r e d a t various locations  85  A m i t o t i c spread comprised o f e l o n g a t e chromosomes, o b t a i n e d from a male N_. hubbsi c o l l e c t e d i n the Wishkah R. d r a i n a g e  86  xii  ACKNOWLEDGEMENTS I am e s p e c i a l l y g r a t e f u l t o Dr. N. J . W i l i m o v s k y , my a d v i s o r , f o r his  continual  critical  g u i d a n c e , s u p p o r t and p a t i e n c e .  analysis of this  committee:  T e c h n i c a l a d v i c e and  paper were p r o v i d e d by t h e members o f my  Dr. N. R. L i l e y ,  Dr. J . D. M c P h a i l , Dr. G. E. E. Scudder  and Dr. A. G. Lewis. S e v e r a l people have p r o v i d e d h e l p o f v a r i o u s k i n d s : Berger, U n i v e r s i t y o f B r i t i s h  Dr. J . D.  Columbia, f a c i l i t i e s f o r Feulgen-DNA  d e n s i t o m e t r y ; Dr. T. Gurney, U n i v e r s i t y o f Utah, f a c i l i t i e s f o r f l u o r o m e t r i c DNA d e t e r m i n a t i o n s ; Dr. F. J . D i l l , U n i v e r s i t y o f B r i t i s h Columbia, use o f a Z e i s s photomicroscope;  Dr. F. Hoppensteaedt,  U n i v e r s i t y o f Utah, a d v i c e on s t a t i s t i c s ; Mr. E. R i d g e s , U n i v e r s i t y o f Utah, access t o t h e UNIVAC 1160 computer; Mr. G. McMines, Q u i n a u l t I n d i a n Nation, permission to c o l l e c t University of British provided e d i t o r i a l  f i s h on I n d i a n l a n d s ; and Mr. S. E. Campana,  Columbia, f i e l d a s s i s t a n c e .  Ms. L. C. A. R o s e n f e l d  a s s i s t a n c e d u r i n g t h e f i n a l stages o f t h i s  L a s t , but by no means l e a s t , Romero f o r t h e t y p i n g o f t h i s  work.  I wish t o express my a p p r e c i a t i o n t o L. J . manuscript.  T h i s i n v e s t i g a t i o n was s u p p o r t e d by a N a t i o n a l S c i e n c e s and E n g i n e e r i n g Research C o u n c i l g r a n t t o N. J . W i l i m o v s k y .  INTRODUCTION Watershed a l t e r a t i o n s r e s u l t i n g from g e o l o g i c a l and c l i m a t i c processes  have been i m p o r t a n t d e t e r m i n a n t s  of f r e s h w a t e r f i s h e s (Myers, 1951; 1981).  One  long-term  f o r the d i s t r i b u t i o n s  D a r l i n g t o n , 1957;  Nelson and  Platnick,  consequence of these d r a i n a g e changes has been t h e  r e s t r i c t i o n o f some s p e c i e s t o s m a l l p a r t s of t h e former these r e l i c t  range.  For  (= e p i b i o t i c ) f i s h e s , r e c e n t p o p u l a t i o n s are of p o t e n t i a l  e v o l u t i o n a r y i n t e r e s t regarding the c o n t r i b u t i o n s of i s o l a t i o n habitat i n determining t h e i r genetic d i f f e r e n t i a t i o n .  and  Perhaps t h e most-  e x t e n s i v e l y s t u d i e d r e l i c t s p e c i e s i n North America a r e t h e  cyprinodonts  t h a t s u r v i v e d t h e p o s t - P l e i s t o c e n e d e s s i c a t i o n o f Death V a l l e y and contiguous  r e g i o n s (see Naiman and S o l t z , 1981).  Considerable  interest  has a l s o been d i r e c t e d towards t h e a s s o c i a t i o n between r e l i c t f i s h e s former 1981).  Pleistocene glacial  r e f u g i a ( e . g . , Franz and  The C h e h a l i s R i v e r d r a i n a g e comprises  Lee, 1976;  and  Trautman,  a former p e r i g l a c i a l  refuge  near t h e t e r m i n a l moraine of t h e l a t e P l e i s t o c e n e Vashon G l a c i e r i n western  Washington, U.S.A. (Heusser,  area i s a f i s h ,  Novumbra hubbsi  p r o b a b l y had a more widespread ( M c P h a i l , 1967;  1964).  Restricted to this  general  S c h u l t z 1929a ( E s o c o i d e i : Umbridae), t h a t d i s t r i b u t i o n i n past g e o l o g i c a l t i m e s  Cavender, 1969).  _N. hubbsi belongs t o a s y s t e m a t i c a l l y -  c o n f u s i n g f a m i l y d i s t r i b u t e d on t h r e e c o n t i n e n t s (Cavender, 1969). il*  hubbsi o c c u r s over a 12000 km  2  range ( F i g . I ) .  S c h u l t z (1929a,b)  d e s c r i b e d t h i s f i s h as having a d i s j u n c t d i s t r i b u t i o n w h o l l y w i t h i n t h e C h e h a l i s R i v e r r e f u g e , but o t h e r c a p t u r e r e c o r d s have i n d i c a t e d a p o s t -  F i g u r e I . Map o f t h e Olympic P e n i n s u l a and a d j a c e n t r e g i o n s showing t h e range o f N. hubbsi (shaded a r e a ) , based on data i n McPhail (1967), Meldrim (1968) and Hagen and Moodie (1979). The l o c a t i o n s o f c o l l e c t i n g s i t e s a r e approximated by s t a r s ( * ) , w i t h t h e e x c e p t i o n o f t h e C o p a l i s R i v e r s i t e , and a c o r r e s p o n d i n g number as f o l l o w s : 1, Q u i n a u l t R i v e r d r a i n a g e ( T a h o l a h ) ; 2, C o p a l i s R i v e r d r a i n a g e ; 3, Conner Creek d r a i n a g e ; 4, c o a s t a l d r a i n a g e ( O y h u t ) ; 5, Humptulips R i v e r d r a i n a g e ; 6, Wishkah R i v e r d r a i n a g e ; 7, C h e h a l i s R i v e r d r a i n a g e ( J u n c t i o n C i t y ) ; 8, Wynoochee R i v e r d r a i n a g e ; 9, Satsop R i v e r d r a i n a g e ; 10, B l a c k R i v e r d r a i n a g e ; 1 1 , Hanaford Creek d r a i n a g e . The C o p a l i s R i v e r c o l l e c t i o n was o b t a i n e d from t h e U n i v e r s i t y o f Washington f i s h c o l l e c t i o n , from which l o c a t i o n a l data was not d e t a i l e d f o r these specimens. See Table 1 f o r map c o - o r d i n a t e s o f and d i r e c t i o n s t o t h e c o l l e c t i n g s i t e s .  kilometers  4  P l e i s t o c e n e d i s p e r s a l i n t o c o n t i g u o u s areas once covered by g l a c i a l ice.  See Heusser  (1964) and McKee (1972) f o r d e t a i l s of t h e Vashon  g l a c i a t i o n i n t h e r e g i o n s c u r r e n t l y i n h a b i t e d by _N. h u b b s i . T h i s s p e c i e s was Fitzgerald  (1957).  f i r s t noted i n t h e Deschutes  McPhail  (1967) and Meldrim  R i v e r d r a i n a g e by  (1968) r e c o r d e d t h i s  i n Olympic c o a s t a l d r a i n a g e s as f a r n o r t h as t h e Queets R i v e r . d u c t i o n s by man  Intro-  are p r o b a b l y r e s p o n s i b l e f o r t h e presence o f _N. hubbsi  even f u r t h e r n o r t h i n Lake Ozette (Wydoski al.  fish  and Whitney, 1979).  Hagen e t  (1979) c o l l e c t e d _N. hubbsi i n P u y a l l u p and Skookum C r e e k s , t h e former  system d r a i n i n g west i n t o s o u t h e r n Puget Sound and t h e l a t t e r d i s c h a r g i n g i n t o t h e same from t h e e a s t near S h e l t o n , Washington.  C e r t a i n marshes  and swamps c o n t a i n e d w i t h i n t h e C h e h a l i s R i v e r system sometimes o v e r f l o w i n t o c o n t i g u o u s d r a i n a g e s d u r i n g p e r i o d s of heavy p r e c i p i t a t i o n f i s h t o invade regions o u t s i d e the peri g l a c i a l Whitney, 1979).  r e f u g e (Wydoski  allowing and  Skookum Creek o r i g i n a t e s i n t h e same area as t h e  C l o q u a l l u m R i v e r , t h e l a t t e r system p a r t of t h e C h e h a l i s R i v e r drainage.  Novumbra from Skookum Creek may  River tributary. Deschutes  have come from t h i s C h e h a l i s  P u y a l l u p Creek f i s h c o u l d have o r i g i n a t e d  R i v e r populations through d i s p e r s a l  from  a c r o s s t h e Puget  Lowlands.  The r e s t r i c t i o n o f _N. hubbsi t o l o t i c peaty waters has been noted s e v e r a l t i m e s ( S c h u l t z 1929a,b; M c P h a i l , 1967; M e l d r i m , 1968; al.  Hagen e t  1979) and such h a b i t a t appears t o be t y p i c a l l y o c c u p i e d by t h e o t h e r  c l o s e r e l a t i v e s ( O s t d i e k and  Nardone, 1959, f o r P a l l i a p e c t o r a l i s Bean;  L e l e k , 1980, f o r Umbra krameri  (Walbaum); Peckham and Dineen, 1957, f o r  JJ. 1 i m i ( K i r t l a n d ) ; Hoese, 1963, f o r ]J_. pygmaea (DeKay)).  Experimental  o b s e r v a t i o n s have i n d i c a t e d t h a t JJ. hubbsi p r e f e r s water w i t h minimal f l o w , l i t t l e o r no d i s s o l v e d s a l t , and reduced l i g h t i n t e n s i t y  (Meldrim,  5 1968), c h a r a c t e r i s t i c s o f the h a b i t a t o c c u p i e d fossil  e v i d e n c e f o r the o c c u p a t i o n  by Novumbra d u r i n g t h e Oligocene The  habitat preference  i t s patchy d i s t r i b u t i o n .  i n nature.  There i s a l s o  o f bogs, swamps or f r e s h w a t e r  (Cavender, 1969).  of_N. hubbsi  i s l i k e l y a major determinant of  S i n c e h a b i t a t s are d i s c o n t i n u o u s ,  g e n e t i c d i f f e r e n t i a t i o n may  marshes  significant  have o c c u r r e d between p o p u l a t i o n s .  The  e x t e n t o f t h i s d i f f e r e n t i a t i o n would depend on p o p u l a t i o n s i z e , gene f l o w and t i m e as w e l l as n a t u r a l s e l e c t i o n (Bush et a l . , 1977; Johnson and M i c k e v i c h , 1977). c l i m a t i c heterogeneity differentiation.  _N. hubbsi  may  Endler,  not have been s u b j e c t e d  The  to  sufficient for significant interpopulational  This f i s h i s found almost e x c l u s i v e l y w i t h i n a s p e c i f i c  h a b i t a t , r a t h e r than a wide v a r i e t y of w a t e r s , over a l i m i t e d area.  1977;  c l i m a t e does not g r e a t l y d i f f e r over the s p e c i e s  geographic  range,  everywhere c h a r a c t e r i z e d by high p r e c i p i t a t i o n d u r i n g t h e c o o l e r months, r e l a t i v e l y dry summers, and m i l d r a r e l y - f r e e z i n g w i n t e r s 1980).  (Ruffner,  C o n s e q u e n t l y , i n t e r r e g i o n a l c l i m a t i c f a c t o r s c o u l d have l i t t l e  i n f l u e n c e upon i n t e r p o p u l a t i o n a l v a r i a t i o n . Some i n f o r m a t i o n i s a v a i l a b l e on i n t r a - and i n t e r p o p u l a t i o n a l v a r i a t i o n i n e x t e r n a l morphology f o r _N. h u b b s i .  Schultz  enumerated e x t e r n a l c h a r a c t e r s on specimens c a p t u r e d l o c a l i t y , a peaty d i t c h between Elma and  r a y s , 6-7;  caudal  11-13; and l a t e r a l  at t h e  type  Satsop about 35 km upstream from  t h e mouth of the C h e h a l i s R i v e r ( F i g . I ) . _N. hubbsi v a r i a t i o n as f o l l o w s :  (1929a)  showed a range o f  number o f p e c t o r a l f i n r a y s , 18-23; p e l v i c f i n  f i n r a y s , 17-22; d o r s a l f i n r a y s , 12-15; anal f i n r a y s , row s c a l e s ,  52-58.  Most specimens examined by S c h u l t z (1929a) were j u v e n i l e s l e s s than 1.5  cm s t a n d a r d  l e n g t h (personal o b s e r v a t i o n ) .  Since c o u n t i n g e r r o r s a r e  6 more l i k e l y t o o c c u r w i t h small specimens (Moyle and Cech, 1982), some c h a r a c t e r s may dark l a t e r a l  have been i n c o r r e c t l y e v a l u a t e d .  S c h u l t z (1929a) r e p o r t e d  b a r r i n g on r e p r o d u c t i v e males but i n d i s t i n c t o r f a i n t  p a t t e r n s on b r e e d i n g f e m a l e s .  A l t h o u g h t h i s was  sexual dimorphism, o t h e r c h a r a c t e r s may  his only reference t o  have d i f f e r e d between s e x e s ,  e s p e c i a l l y i f t h e s e were somehow i m p o r t a n t t o sexual r o l e s d u r i n g r e p r o d u c t i on. il«  hubbsi does not possess  a lateral  t h e p o s i t i o n o c c u p i e d by a t y p i c a l  lateral  l i n e but has pored s c a l e s i n l i n e ( S c h u l t z , 1929a).  Meldrim  (1968) l o o k e d at geographic  v a r i a t i o n i n t h e number of t h e s e  lateral  row s c a l e s . _N. hubbsi from t h e C h e h a l i s R i v e r d r a i n a g e had 50-59  scales.  Specimens from t h e Q u i n a u l t R i v e r system, an Olympic c o a s t a l  drainage  ( F i g . 1 ) , had l e s s e r numbers, 42-48, w h i l e f i s h from swamps i n  t h e Humptulips R i v e r d r a i n a g e had 43-58. a cline in lateral inland l o c a l i t i e s .  These data may  be i n d i c a t i v e of  s c a l e numbers i n c r e a s i n g from t h e P a c i f i c c o a s t t o S i n c e s c a l e counts were most v a r i a b l e i n t h e  Humptulips R i v e r c o l l e c t i o n , two  Novumbra morphs may  have been i n d i c a t e d  w i t h t h e i r zone of i n t e r g r a d a t i o n w i t h i n t h e Humptulips R i v e r r e g i o n . The number of specimens examined by Meldrim 100 i n d i v i d u a l s .  (1968) was  fewer than  The f i s h were not d e p o s i t e d i n a museum c o l l e c t i o n , so  t h e study m a t e r i a l i s not r e a d i l y a v a i l a b l e f o r v e r i f i c a t i o n .  Adults of  both sexes as w e l l as j u v e n i l e s were enumerated t o g e t h e r , so s e x u a l l y d i m o r p h i c o r o n t o g e n e t i c f a c t o r s were not c o n s i d e r e d . (1968) d i d not d e f i n e how  Since  Meldrim  he d i d h i s m e r i s t i c c o u n t s , v e r i f i c a t i o n of h i s  r e s u l t s might be d i f f i c u l t even i f h i s c o l l e c t i o n s were a v a i l a b l e . Meldrim  (1968) s t a t e d t h a t C h e h a l i s R i v e r _N. hubbsi  had b a r r e d  m a r k i n g s , w h i l e t h o s e from t h e Q u i n a u l t R i v e r d r a i n a g e possessed  lateral mottled  •7 patterns.  T h i s o b s e r v a t i o n was n e i t h e r d e f i n e d nor i l l u s t r a t e d .  A l t h o u g h t h e r e e x i s t many i c h t h y o l o g i c a l  s t u d i e s on  geographic  v a r i a t i o n i n e x t e r n a l morphology, few i n v e s t i g a t o r s have examined chromosomal s t a t e s a c r o s s a s p e c i e s range.  Chromosomal  t h e o r g a n i z a t i o n of t h e chromosome complement.  state refers to  In t h i s s t u d y , t h e term  i s r e s t r i c t e d t o chromosome numbers and c e n t r o m e r i c l o c a t i o n s determined by means o f Giemsa o r o r c e i n s t a i n i n g .  Methods a r e a v a i l a b l e , e s p e c i a l l y  w i t h chromosomes from h i g h e r v e r t e b r a t e s (Hsu, 1973; B l a x h a l l , 1975), f o r l o o k i n g a t v a r i a t i o n i n intrachromosomal o r g a n i z i n g and h e t e r o c h r o m a t i c sequences.  f e a t u r e s such as n u c l e o l a r  r e g i o n s as w e l l as s p e c i f i c genes o r DNA  See Swanson e t a l . (1981) f o r a d i s c u s s i o n on chromosomal  analyses i n systematic b i o l o g y . The d i s j u n c t d i s t r i b u t i o n o f _N. hubbsi  c o u l d mean t h a t t h i s  fish  o c c u r s i n s m a l l b r e e d i n g u n i t s , a c r i t e r i o n c o n s i d e r e d i m p o r t a n t by some i n v e s t i g a t o r s f o r the establishment of i n t e r p o p u l a t i o n a l polymorphisms (Bush, 1975; W i l s o n e t a l . , 1975). o f salmonids  by Thorgaard  chromosomal  The c y t o g e n e t i c study  (1978) i n v o l v e d k a r y o t y p i c a n a l y s e s on  animals  from A l a s k a t o C a l i f o r n i a , perhaps t h e most comprehensive e x a m i n a t i o n i n t r a s p e c i f i c chromosomal d i v e r s i t y i n f i s h e s t o d a t e . (1978, 1979) have l o o k e d a t geographic cyprinodontids.  of  B l a c k and Howell  chromosomal v a r i a t i o n i n  The above r e s e a r c h e r s have shown t h a t chromosomal s t a t e s  can vary between p o p u l a t i o n s o f c o n s p e c i f i c s .  Chromosomal  f i s h e s has u s u a l l y been a s s o c i a t e d w i t h geographic  variation i n  isolation  (Gold e t  a l . , 1977; Black and H o w e l l , 1978, B e r t o l l o e t a l . , 1979, LeGrand and Cavender, 1980) a l t h o u g h environmental determinants  f a c t o r s have a l s o been c o n s i d e r e d  o f chromosomal s t a t e s ( N i k o l s k y and V a s i l y e v , 1974;  N i k o l s k y , 1976).  •8 Most c y t o g e n e t i c s t u d i e s on f i s h e s have i n v o l v e d few chromosome spreads  from even fewer animals u s u a l l y c a p t u r e d at one l o c a t i o n  (e.g.,  most r e f e r e n c e s l i s t e d i n Gold e t a l . , 1980), p r o b a b l y under t h e assumption t h a t chromosomal s t a t e s are f i x e d w i t h i n s p e c i e s 1978).  (White,  The o n l y p u b l i s h e d chromosomal a n a l y s i s on _N. hubbsi was  w i t h s i x m i t o t i c spreads locality  from two specimens caught at an u n s p e c i f i e d  (Beamish e t a l . , 1971).  interpopulational  Consequently,  nobody has l o o k e d a t  v a r i a t i o n i n chromosomal s t a t e s i n t h i s s p e c i e s .  modal d i p l o i d number f or _N. hubbsi was chromosome p a i r s  done  The  r e p o r t e d as 48 chromosomes o r  24  (Beamish et a l . , 1971), but t h i s s h o u l d be c o n s i d e r e d as  t e n t a t i v e f o r t h e s p e c i e s as a whole u n t i l  i n d i v i d u a l s from s e v e r a l  p o p u l a t i o n s are examined. Beamish et a l . (1971) determined w e l l as o t h e r c l o s e r e l a t i v e s . g r o u p i n g s , w i t h DNA f o r _N. hubbsi  v a l u e s f or JN.  These are p r e s e n t e d  contents expressed  ( w i t h an a b s o l u t e DNA  di p i o i d nucleus):  n u c l e a r DNA  hubbsi  here i n two  as m u l t i p l e s of t h e d e t e r m i n a t i o n  c o n t e n t of 2.08  pg  (picograms)/-  P a l l i a p e c t o r a l i s Bean and Esox spp. w i t h 1.08-1.31  t i m e s ; and Umbra spp. w i t h 2.32  - 2.42  times.  The ONA  values f o r _ N .  h u b b s i , JJ. p e c t o r a l i s and Esox spp. were 40-56% of t h o s e determined salmonids  as  by Vendrely  (1953), Knobloch e t a l . (1957) and  (1968), perhaps evidence f o r t h e h y p o t h e s i z e d t e t r a p l o i d a l t h e salmonid genome from t h a t of a d i p l o i d s a l m o n i f o r m  for  Hinegardner d e r i v a t i o n of  ancestor  (Zenzes  and V o i c u l e s c u , 1976). The a c c u r a c y of t h e e s o c o i d DNA (1971) i s q u e s t i o n a b l e . i t may  d e t e r m i n a t i o n s by Beamish e t a l .  Only Feulgen-DNA methodology was  have been b e t t e r t o c o r r e l a t e o t h e r DNA  w i t h Feulgen-DNA cytophotometry  used, a l t h o u g h  quantification  because of d i f f i c u l t i e s i n t h e  techniques  9-  s t a n d a r d i z a t i o n o f t h e l a t t e r (McLeish and S u n d e r l a n d , 1961; M i k s c h e , 1971).  A l s o t h e sample s i z e s were s m a l l .  F o r example, o n l y two _N.  hubbsi from a s i n g l e u n s p e c i f i e d l o c a l i t y were examined. A l t h o u g h t h e DNA constancy p r i n c i p l e ( M i r s k y and and R i s , 1949) was l i k e l y assumed i n a s c e r t a i n i n g genomic c o n t e n t s , l i t e r a t u r e i s a v a i l a b l e showing t h a t DNA v a l u e s may d i f f e r between p o p u l a t i o n s o f c o n s p e c i f i c s . The l a r g e s t d i f f e r e n c e s i n n u c l e i c a c i d c o n t e n t a r e p l o i d a l  ones and  e x t e r n a l morphology o r s i z e may not be u s a b l e f o r d i s c e r n i n g between individuals with different ploidal  s t a t e s (e.g., t h e races of c o b i t i d s  s t u d i e d by S e z a k i and K o b a y a s h i , 1978).  Ploidal  d i f f e r e n c e s are r e a d i l y  i d e n t i f i e d by c o u n t i n g chromosomes s i n c e complements would c o n t a i n m u l t i p l e s o f t h e h a p l o i d chromosome number. Sherwood and P a t t o n (1982) found e x t e n s i v e i n t e r p o p u l a t i o n a l  DNA  v a r i a t i o n i n t h e r o d e n t , Thomomys b o t t a e (Eydoux and G e r v a i s ) , a l t h o u g h specimens had s i m i l a r k a r y o t y p e s .  DNA c o n t e n t s were found t o d i f f e r by  as much as 35%, i n d i c a t i n g t h a t d i f f e r i n g n u c l e i c a c i d c o n t e n t s may not be always d e t e c t a b l e by l o o k i n g a t chromosome s p r e a d s .  No f i s h e s o t h e r  than some d i p l o i d - p o l y p l o i d forms ( S e z a k i e t a l . , 1977) have been examined f o r g e o g r a p h i c v a r i a t i o n i n DNA c o n t e n t . T h i s s t u d y i n t e n d s t o examine g e o g r a p h i c v a r i a t i o n i n _ N . h u b b s i , addressing the following questions: (a)  How does t h e e x t e r n a l phenotype, s p e c i f i c a l l y  c h a r a c t e r s , v a r y a c r o s s t h e range? s p e c i e s i n t h e manner suggested  meristic  Is _N. hubbsi a polymorphic  by Meldrim's  (1968) d a t a ?  (b)  Does chromosome morphology v a r y ?  (c)  Do i n d i v i d u a l s from d i f f e r e n t p o p u l a t i o n s have d i f f e r e n t  amounts o f DNA?  10 The analyses  r e s u l t s o f t h e e x t e r n a l m e r i s t i c , chromosomal, and n u c l e a r will  DNA  be c o r r e l a t e d w i t h g e o l o g i c a l and e c o l o g i c a l i n f o r m a t i o n t o  i n f e r zoogeographical  and s e l e c t i o n a l h i s t o r i e s .  As emphasized by  C h e r n o f f (1982), such hypotheses a r e a n e c e s s a r y p r e l i m i n a r y t o understanding  character  variation.  Land use p r a c t i c e s have e l i m i n a t e d much  Novumbra h a b i t a t (Wydosky and Whitney, 1979) and such a c t i v i t i e s have r e s u l t e d i n t h e e x t i n c t i o n o f r e l i c t f i s h e s elsewhere ( P i s t e r , 1981). C o n s e q u e n t l y , b a s e l i n e data on p o p u l a t i o n information  v a r i a t i o n i s important  f o r f u t u r e p r o t e c t i o n and management o f N.  hubbsi.  11  MATERIALS AND METHODS  Sampling  localities  _N. hubbsi were c o l l e c t e d from a major p a r t o f t h e i r range, w i t h i n t h e C h e h a l i s R i v e r d r a i n a g e as w e l l as Olympic c o a s t a l ones.  The  s a m p l i n g s i t e s a r e shown i n F i g . I and map c o - o r d i n a t e s o f as w e l l as d i r e c t i o n s t o each s i t e a r e g i v e n i n Table 1.  C l a s s i f i c a t i o n o f sampling  localities  Each c o l l e c t i n g s i t e was c a t e g o r i z e d a c c o r d i n g t o i t s source o f water.  Water b o d i e s kept f i l l e d o n l y by p r e c i p i t a t i o n and groundwater  p e r c o l a t i o n were termed c l o s e d ( l a n d l o c k e d ) , w h i l e t h o s e f e d by were c l a s s i f i e d as open ( d r a i n e d ) .  streams  S u r f a c e areas were c a l c u l a t e d f o r a l l  s a m p l i n g s i t e s by p a c i n g o f f s h o r e l i n e p e r i m e t e r s t o o b t a i n c i r c u m f e r e n c e s from which areas were c a l c u l a t e d and/or by r e f e r e n c e t o t o p o g r a p h i c maps.  T h i s measure was c o n s i d e r e d i n d i c a t i v e o f h a b i t a t s i z e  s i n c e swampy h a b i t a t s were m o s t l y f l a t and s h a l l o w .  S i n c e _N. hubbsi i s  u s u a l l y found i n water l e s s than 1.5 m deep (Meldrim, 1968), t h i s was c o n s i d e r e d t o have had a p o t e n t i a l particular  Physical  fish  presence anywhere w i t h i n a  site.  and chemical d e t e r m i n a t i o n s on t h e water  P h y s i c a l and chemical  d e t e r m i n a t i o n s o f t h e water were made upon  each v i s i t t o a l l c o l l e c t i n g s i t e s .  Temperature was a s c e r t a i n e d w i t h a  s t a n d a r d c e n t i g r a d e thermometer, pH w i t h Hydrion t e s t p a p e r s ,  dissolved  oxygen u s i n g t h e W i n k l e r method, and carbon d i o x i d e by means o f a Hach  T a b l e 1. Drainage l o c a t i o n s , map c o - o r d i n a t e s and s i t e d i r e c t i o n s f o r H. hubbsi c o l l e c t i n g s i t e s . The names f o r c o l l e c t i n g s i t e s a r e those used i n t h e t e x t . Drainages marked w i t h a s t e r i s k s (*) a r e t r i b u t a r i e s o f t h e C h e h a l i s R i v e r , t h e remaining ones d i s c h a r g e d i r e c t l y i n t o t h e P a c i f i c Ocean. L a t i t u d e s and l o n g i t u d e s were o b t a i n e d from U. S. G e o l o g i c a l Survey 15-minute S e r i e s t o p o g r a p h i c maps. Col 1 e c t i ng Site  Drainage  Latitude  Longitude  Taholah  Q u i n a u l t R.  47°29'50"  124°17'35"  0.4 km west o f Q u i n a u l t Indian Nation headquarters a t T a h o l a h , swamp a d j a c e n t t o westernmost housing complex.  Copal i s  C o p a l i s R.  47°11'05" 47°15'00"  124°07'30" 124°01'45"  Specimens from t h e C o p a l i s R. d r a i n a g e were c o l l e c t e d i n 1948 and a r e c u r a t e d by t h e U n i v e r s i t y o f Washington C o l l e g e o f F i s h e r i e s . L o c a l i t y i n f o r m a t i o n i s a p p a r e n t l y l o s t . The l i s t e d l a t i t u d e s and l o n g i t u d e s comprise t h e geographic area covered by t h e C o p a l i s R.  Conner Creek  Conner Creek  47°03'48"  124°09'15"  A p p r o x i m a t e l y 6 km upstream from t h e mouth o f Conner Creek, s i t e d e s t r o y e d d u r i n g t h e c o n s t r u c t i o n o f a housing p r o j e c t (Wydoski and Whitney, 1979).  Oyhut  Coastal  47°01'45"  124 09'10"  1.1 km s o u t h o f t h e e n t r a n c e t o Ocean Shores S t a t e Park on highway t o Oyhut, swamp on t h e east s i d e o f t h e road where highway a b r u p t l y t u r n s west.  Humptul i ps  Humptulips R.  47°03'45"  124°01'15"  Burrows Road 1 km n o r t h o f i t s i n t e r s e c t i o n w i t h S t a t e Route 9C, swamp on t h e e a s t s i d e o f t h e road.  o  Directions to collecting  site  T a b l e 1.  (Continued)  Collecting Site  Drainage  Latitude  Longitude  Directions t o collecting s i t e  Wishkah  Wishkah R.  47°00'11"  123°48'12"  1.1 km n o r t h o f t h e e n t r a n c e t o Fern H i l l Cemetery on East Wishkah Road, where stream c r o s s e s under t h e highway, swamp on t h e e a s t side o f t h e road.  Junction City  C h e h a l i s R.  46°58'43"  123°45'58"  0.6 km s o u t h e a s t o f t h e b r i d g e a c r o s s Northern E l l i o t Slough on t h e highway t o J u n c t i o n C i t y , marshy a r e a on t h e west s i d e o f t h e r o a d .  Wynoochee  Wynoochee R.*  46°59'20"  123°39'04"  2.1 km n o r t h o f U. S. Route 410 on West Wynoochee Road, where stream c r o s s e s under t h e r o a d , swamp c o n t i g u o u s t o t h e west s i d e o f t h e hi g hw ay.  Satsop  Satsop R.*  47°06'40"  123°24'45"  13 km n o r t h e a s t o f Schaefer S t a t e Park and 2 km n o r t h e a s t o f t h e Matthews homestead on M a t l o c k S h e l t o n Road, f l o o d e d r e g i o n a l o n g Dry Run Creek on t h e northwest s i d e o f t h e highway.  Black R i v e r  Black R.*  46°52'30"  123°01'20"  2.2 km s o u t h o f L i t t l e Rock on S t a t e Route 1M, marshy s l o u g h 40 m west o f t h e highway.  Hanaford Creek  Hanaford  46°45'03"  122°55'08"  5.6 km e a s t o f S t a t e Route 507 on Hanaford V a l l e y Road, a p p r o x i m a t e l y 100 m s o u t h o f t h e highway, an oxbow pond c o n t i g u o u s t o Hanaford Creek.  Creek*  14  kit.  D i s s o l v e d s a l t c o n c e n t r a t i o n was  Instrument  a s c e r t a i n e d w i t h a Yellow  Springs  Model 33 c o n d u c t i v i t y meter.  Methods o f c o l l e c t i o n F i s h were c a p t u r e d w i t h a 3-m as metal  minnow t r a p s .  l o n g 6.4-mm mesh beach s e i n e as w e l l  The l a t t e r were u s u a l l y b a i t e d w i t h c a t food  p l a c e d i n the water o v e r n i g h t .  and  Both c a p t u r e methods were used at a l l  s i t e s ; however, most specimens were o b t a i n e d w i t h t r a p s s i n c e many s i t e s were c l u t t e r e d w i t h sunken d e b r i s t h a t tended t o e n t a n g l e and t e a r t h e seine.  A finely-meshed  d i p n e t w i t h a 30 cm opening was  one o c c a s i o n at each l o c a t i o n . November 1978  used on at l e a s t  A l l _N. hubbsi were caught between  and December 1979.  Specimens from Conner Creek and  the  C o p a l i s R i v e r were borrowed from t h e f i s h c o l l e c t i o n at t h e C o l l e g e o f F i s h e r i e s of the U n i v e r s i t y o f Washington at S e a t t l e .  Morphomeristic Captured  determinations f i s h were f i x e d i n 10% b o r a t e - b u f f e r e d f o r m a l i n f o r one  week, then r i n s e d i n r u n n i n g t a p water f o r 6 h, d r a i n e d t o remove excess m o i s t u r e , and p l a c e d i n 37.5% 10 animals  isopropanol p r e s e r v a t i v e .  A p i l o t study  had shown t h a t w e i g h t - l o s s d i d not s t a b i l i z e u n t i l  in preservative.  Consequently,  after 8  on wks  a l l samples were s t o r e d f o r at l e a s t 60 d  p r i o r t o measurement o f s t a n d a r d l e n g t h s t o m i n i m i z e  v a r i a t i o n due  post mortem changes i n p r e s e r v a t i v e .  morphomeristic  d e t e r m i n a t i o n s , o t h e r than C o p a l i s and  F i s h used f o r  to  Conner Creek ones, were d e p o s i t e d  i n t h e f i s h c o l l e c t i o n o f t h e U n i v e r s i t y of B r i t i s h Columbia. With t h e e x c e p t i o n of the l a t e r a l  bar count on m a l e s ,  morphomeristic  d e t e r m i n a t i o n s were made a c c o r d i n g t o the methods of Hubbs and (1964).  Measurements were o b t a i n e d u s i n g a H e l i o s d i a l  Lagler  c a l i p e r and  taken  15 t o t h e n e a r e s t 0.1 and  mm;  t h e s e were t a k e n t h r e e times on t h e same specimen  averaged t o g i v e a s i n g l e v a l u e .  A l l m e r i s t i c counts were made under  a d i s s e c t i n g m i c r o s c o p e , w i t h each count redone at l e a s t once t o a s s u r e accuracy. 1.  Descriptions  of the c h a r a c t e r s  Standard l e n g t h  t o t h e end  of the hypural  through the 2.  (SL):  used f o l l o w .  t h e measurement from t h e t i p of t h e  p l a t e , t h e l a t t e r i d e n t i f i e d by p a s s i n g  L a t e r a l s c a l e row:  by pored s c a l e s .  the number of s c a l e s a l o n g a l i n e i n the  The  count t e r m i n a t e s at t h e end  b e g i n s at the upper o r i g i n of t h e o p e r c u l a r 3.  Scales  above t h e l a t e r a l  above t h e l a t e r a l counted down and  s c a l e row,  s c a l e row:  Scales  below the l a t e r a l  up and  s c a l e row,  row  plate  opening.  f i n and  t h e count t e r m i n a t e d w i t h  the number of s c a l e rows  from the o r i g i n of t h e anal  s c a l e i m m e d i a t e l y below t h e l a t e r a l Scale  of t h e hypural  scale.  s c a l e row:  f o r w a r d f o l l o w i n g the s c a l e row,  5.  row  recognized  t h e number of s c a l e rows  back f o l l o w i n g t h e s c a l e row,  below t h e l a t e r a l  l i n e and  from the o r i g i n of t h e d o r s a l  t h e s c a l e i m m e d i a t e l y b e f o r e the l a t e r a l 4.  light  specimen.  p o s i t i o n t h a t would be o c c u p i e d by a t y p i c a l l a t e r a l  and  snout  f i n and  t h e count t e r m i n a t e d w i t h  counted the  scale.  rows b e f o r e t h e d o r s a l f i n o r i g i n :  rows from t h e upper o r i g i n of the o p e r c u l a r  the number of  scale  opening t o the o r i g i n of  the  dorsal f i n . 6. p l u s one  Dorsal  and  anal  unbranched one,  f i n rays:  t h e count i n c l u d i n g a l l branched rays  s i n c e o n l y a s i n g l e unbranched ray reaches  o u t e r margin of e i t h e r f i n ; the l a s t ray may but i s s t i l l 7.  counted as o n l y  Caudal f i n rays:  the  be d i v i d e d t h r o u g h i t s base  one.  the count of a l l p r i n c i p a l  r a y s , the number of  16  branched rays p l u s two unbranched ones. 8.  P e c t o r a l and p e l v i c f i n rays:  t h e count o f a l l r a y s , i n c l u d i n g  t h e s m a l l e s t ones v i s i b l e o n l y w i t h a d i s s e c t i n g m i c r o s c o p e . 9.  L a t e r a l bars on males:  t h e count o f t h e prominent dark  lateral  bars s t a r t i n g w i t h t h e f i r s t o b v i o u s one p o s t e r i o r t o t h e upper o r i g i n o f t h e o p e r c u l a r o p e n i n g , enumerated a l o n g t h e l a t e r a l  row and t e r m i n a t i n g  w i t h t h e l a s t complete b a r b e f o r e t h e hypural p l a t e . A n a l y s i s o f m e r i s t i c data Whether o r not a c h a r a c t e r had been s e x u a l l y - d i m o r p h i c was determined w i t h Chi-square  t e s t s on m e r i s t i c c h a r a c t e r f r e q u e n c i e s .  If  sexes a t even one l o c a l i t y d i f f e r e d a t t h e p = 0.05 l e v e l , then t h e r e l e v a n t parameter was f u r t h e r t e s t e d s e p a r a t e l y f o r each sex f o r a l l populations.  Variances  o f t h e raw data were u s u a l l y heterogeneous as  checked w i t h B a r t l e t t ' s t e s t , so base-10 l o g a r i t h m i c c o n v e r s i o n s made p r i o r t o p a r a m e t r i c c h a r a c t e r s and s t a n d a r d  analyses.  C o r r e l a t i o n analyses  were  between m e r i s t i c  l e n g t h suggested l i t t l e o r no f u n c t i o n a l  r e l a t i o n s h i p s (Table 2 ) , so each m e r i s t i c parameter was e v a l u a t e d by i t s e l f and not i n r e l a t i o n t o l e n g t h . For c h a r a c t e r s determined t o have been s e x u a l l y - d i m o r p h i c , mean counts f o r males and females w i t h i n each p o p u l a t i o n were compared u s i n g t-tests. variance  Geographic comparisons were f i r s t made w i t h a n a l y s i s o f (ANOVA).  ANOVA i s robust enough t o operate w e l l even w i t h  considerable heterogeneity ( Z a r , 1974).  Although  o f v a r i a n c e s and d e p a r t u r e s  from n o r m a l i t y  v a r i a n c e s may not be e q u a l , a c c e p t a n c e o f ANOVA F-  v a l u e s i s j u s t i f i e d by concordance w i t h t h e Welch and Brown-Forsythe tests  (Brown and F o r s y t h e , 1974).  The Welch t e s t as w e l l as Brown-  17  Table 2. C o r r e l a t i o n s between m e r i s t i c c h a r a c t e r s t a t e s and s t a n d a r d l e n g t h f o r N. h u b b s i . C o l l e c t i o n s from each sampled l o c a l i t y were e v a l u a t e d i n d i v i d u a l l y y i e l d i n g t h e l i s t e d range of c o r r e l a t i o n s . The m e r i s t i c c h a r a c t e r s used i n t h i s study d i d not appear t o be i n f l u e n c e d by s t a n d a r d l e n g t h , a measure o f s i z e .  Overal1 Correl ation  _N  Range of Correlations  Dorsal f i n rays i n males  160  -0.29  -  0.45  0.23  Dorsal f i n rays i n females  149  -0.33  -  0.48  0.08  Anal f i n rays i n males  160  -0.38  -  0.47  0.21  Anal f i n rays i n females  149  -0.39  -  0.43  0.10  Caudal  306  -0.05  -  0.23  0.08  P e c t o r a l f i n rays  248  -0.39  -  0.36  0.01  P e l v i c f i n rays  319  -0.42  -  0.12  -0.09  -0.01  -  0.40  0.08  0.01  -  0.30  0.03  -0.09  -  0.09  0.03  -0.07  -  0.10  -0.02  -0.32  -  0.48  0.04  Meristic character  f i n rays  S c a l e rows b e f o r e dorsal f i n o r i g i n  307  Lateral  307  row s c a l e s  S c a l e s above l a t e r a l  row  303  S c a l e s below l a t e r a l  row  208  Lateral  bars i n males  207  18 " F o r s y t h e t e s t s a r e e s s e n t i a l l y ANOVA's w i t h o u t t h e assumption o f equal variance. variance  Each ANOVA i s p r e s e n t e d w i t h t h e Levene t e s t f o r equal i n turn followed  by t h e Welch and Brown-Forsythe t e s t s .  If the  F - v a l u e was s i g n i f i c a n t , then i t was expected t h a t t h e l a t t e r two s h o u l d be a l s o f o r t h e ANOVA F-value t o be a c c e p t e d . C o r r e l a t i o n a n a l y s i s , t h e Levene t e s t f o r equal v a r i a n c e  as w e l l as  the ANOVA, Welch t e s t and Brown-Forsythe t e s t were done on t h e U n i v e r s i t y of Utah Univac 1160 computer, u t i l i z i n g t h e BMDP-81 s o f t w a r e package (Dixon e t a l . , 1981).  A p o s t e r i o r i comparisons were made w i t h t h e  Student-Newman-Keul m u l t i p l e range t e s t f o r unequal sample s i z e s (SNK) and  S c h e f f e ' s m u l t i p l e c o n t r a s t s t e s t (SMC) as o u t l i n e d by Sokal and  Rohlf  (1969).  SMC i s not as powerful f o r p a i r w i s e comparisons as SNK  ( Z a r , 1974); c o n s e q u e n t l y , a p p l i c a t i o n o f t h e former was r e s t r i c t e d t o s e l e c t e d cases f o r which i t was e s p e c i a l l y s u i t a b l e , m u l t i p l e  contrasts.  Chromosome methodology M e i o t i c and m i t o t i c chromosomes were o b t a i n e d of t h e Kligerman and Bloom (1977) t e c h n i q u e . i n j e c t e d w i t h 20  g c o l c h i c i n e / g body w e i g h t .  as a 0.04% s o l u t i o n i n C o r t l a n d ' s containing  0.25% c a l c i u m  chloride.  using a m o d i f i c a t i o n  F i s h were i n t r a p e r i t o n e a l l y The c o l c h i c i n e was made up  p h y s i o l o g i c a l s a l i n e (Wolf, 1963) This a d d i t i o n of calcium  Subrahmanyam (1969), who used t h i s i o n t o s t i m u l a t e c e l l counter colchicine-induced were p l a c e d f o r 6 h. and, and  c o n t r a c t i o n o f chromosomes.  i n a well-aerated  container  followed  d i v i s i o n and Injected  fish  a t room t e m p e r a t u r e (20 - 22°C)  Specimens were s a c r i f i c e d by t r i c a i n e m e t h a n o s u l f o n a t e overdose  as a p p r o p r i a t e ,  gill  t e s t e s were removed.  a r c h e s , f i n edges, k i d n e y , i n t e s t i n a l  tract,  The e x t i r p a t e d t i s s u e was g e n t l y and q u i c k l y  19 <•• washed w i t h 0.4% vial  h y p o t o n i c KC1 s o l u t i o n .  T i s s u e s were then p l a c e d i n a  w i t h a p p r o x i m a t e l y 15 t i m e s t h e i r volume of 0.4%  20 - 30 min a t room t e m p e r a t u r e .  KC1  solution for  A f t e r removal o f as much h y p o t o n i c  s o l u t i o n as p o s s i b l e , t h e sample was f i x e d i n t h e same v i a l .  Fixation  e n t a i l e d at l e a s t 3 changes of f r e s h l y - p r e p a r e d 3:1 anhydrous  methanol-  a c e t i c a c i d at 4°C f o r at l e a s t 30 min each.  T i s s u e s were u s u a l l y  p r o c e s s e d onto s l i d e s i m m e d i a t e l y a f t e r t h i s f i x a t i o n , but t h e y were sometimes s t o r e d i n t h e r e f r i g e r a t o r f o r up t o 72 h. All  s l i d e s were c l e a n s e d w i t h A l c o n e x , washed w i t h hot w a t e r , and  r i n s e d thoroughly with d i s t i l l e d water. methanol  until  needed.  F i n i s h e d s l i d e s were s t o r e d i n  S l i d e s were wiped d r y w i t h a Kimwipe and p l a c e d  on a s l i d e warmer heated t o 40 - 45°C.  A p i e c e of t i s s u e was  removed  from t h e f i x a t i v e , touched t o a c l e a n paper towel t o remove excess f i x a t i v e , and p l a c e d i n t o t h e w e l l  of a Boerner s l i d e .  Four t o s i x drops  o f 50% a c e t i c a c i d were q u i c k l y added and t h e t i s s u e g e n t l y minced f o r a p p r o x i m a t e l y 1 min t o form a c e l l  slurry.  A non-heparinized  m i c r o h e m a t o c r i t tube ( S c i e n t i f i c P r o d u c t s , B4416-1), mounted t o a tuberculin  s y r i n g e f o r c o n t r o l l e d s u c t i o n , was f i l l e d w i t h  s u s p e n s i o n and e x p e l l e d onto a warmed s l i d e .  cell  The s l u r r y was a l l o w e d t o  remain on t h e s l i d e f o r 15 - 20 sec and then withdrawn back i n t o t h e c a p i l l a r y tube.  A c i r c l e o f c e l l s would remain on t h e s l i d e .  f i l l e d with cell  s u s p e n s i o n s l u r r y was s u f f i c i e n t f o r 3-4  One t u b e  circles.  S l i d e s were a l l o w e d t o d r y , p l a c e d i n a d u s t - f r e e s l i d e box, and s t o r e d i n a c o o l , dry l o c a t i o n f o r 72 h b e f o r e s t a i n i n g . days seemed t o improve s t a i n i n g  A g i n g s l i d e s f o r a few  quality.  S l i d e s were s t a i n e d i n f r e s h 10% Giemsa (Gurr Improved R66) p r e p a r e d i n M/15  Sorensen b u f f e r a t pH 6.8.  S t a i n was always f i l t e r e d b e f o r e  20 use.  S t a i n e d s l i d e s were r i n s e d w i t h d i s t i l l e d w a t e r , b l o t t e d w i t h  b i b u l o u s paper, a i r d r i e d , and p l a c e d i n x y l e n e f o r at l e a s t 15 S l i d e s were mounted i n Permount w i t h a No.  min.  1 coverslip.  F i n i s h e d s l i d e s were observed under a Z e i s s Photomicroscope I I equipped w i t h a 100 W mercury vapor l i g h t s o u r c e . t h r o u g h a 100X Technical  Photographs were taken  phase o b j e c t i v e and a green substage f i l t e r w i t h Kodak  Pan 2415  f i l m ASA  50.  F i l m was  and p r i n t e d on Kodak paper u s i n g Dektol  developed i n Kodak D19 s o l u t i o n developer.  A f t e r p r e l i m i n a r y s o r t i n g of those e n l a r g e d photographs i n which chromosomes were n e i t h e r e x c e s s i v e l y o v e r l a p p i n g nor c o n t r a c t e d , each chromosome was  measured.  Quantitative characterizations involved  measurements of the r e l a t i v e l e n g t h ( e x p r e s s s e d  as percent  l e n g t h o f d i p l o i d complement) and the arm  ( s h o r t arm/long arm  100). 1/60  ratio  of t o t a l X  A l l measurements were taken w i t h c a l i p e r s and a r u l e r graduated i n inch units.  The t e r m i n o l o g y  of Levan et a l . (1964) was  used f o r  d i s c u s s i n g chromosome morphology. Feulgen-DNA  determinations  Nucleated  e r y t h r o c y t e s were chosen s i n c e they are d i p l o i d ,  i n s i z e and shape, and  r e a d i l y a v a i l a b l e as s i n g l e c e l l s .  Blood  uniform was  o b t a i n e d by s a c r i f i c i n g t h e f i s h and d r a i n i n g the heart w i t h a h e p a r i n i z e d c a p i l l a r y tube ( S c i e n t i f i c P r o d u c t s , B4416-2). 2 h b e f o r e f i s h were t o be p r o c e s s e d ,  b l o o d was  No more than  a l s o taken from a Rhode  I s l a n d Red c h i c k e n t h r o u g h a wing v e i n u s i n g a h e p a r i n i z e d tube and t h e r e s u l t a n t sample s t o r e d i n a r e f r i g e r a t o r u n t i l  Vacutainer needed.  Clean dry s l i d e s were d i v i d e d i n t o 2 s e c t i o n s w i t h a diamond p e n c i l . Blood was  smeared t h i n l y onto t h e s l i d e s , one s e c t i o n r e c e i v i n g f i s h  21 e r y t h r o c y t e s and t h e o t h e r c h i c k e n b l o o d as a r e f e r e n c e .  The smears were  a i r - d r i e d f o r 5 min and f i x e d a c c o r d i n g t o t h e method o f Mizuno and MacGregor (1974).  S l i d e s were p l a c e d f o r 15 min i n f r e s h l y - m i x e d 3:1  a b s o l u t e e t h a n o l - a c e t i c a c i d a t 4°C and then passed through two 5 min soaks i n 95% e t h a n o l and a i r - d r i e d . S l i d e s were wetted w i t h d i s t i l l e d water and h y d r o l y z e d i n 5N h y d r o c h l o r i c a c i d a t 20°C f o r 40 m i n , s i n c e DNA l o s s s h o u l d have been minimized  under t h e s e c o n d i t i o n s ( D e i t c h e t a l . , 1968; Murgatroyd, 1968;  Vahs, 1973). staining.  The method o f Mizuno and MacGregor (1974) was f o l l o w e d f o r  A f t e r washing t h e s l i d e s i n t h r e e 5 min changes o f d i s t i l l e d  w a t e r , t h e y were s t a i n e d i n S c h i f f ' s reagent a t 23°C i n t h e dark f o r 90 min.  The s l i d e s were then washed i n t h r e e changes o f 0.5% sodium  m e t a b i s u l f i t e f o r 2 min e a c h , and then passed t h r o u g h 5 min soaks i n d i s t i l l e d w a t e r , 70% e t h a n o l , 95% e t h a n o l , a b s o l u t e e t h a n o l , and xylene.  Smears were mounted i n C a r l Z e i s s immersion o i l (nD = 1.515)  w i t h a No. 1 c o v e r s l i p . Two-area cytophotometry microspectrophotometer  o f s t a i n e d n u c l e i was done w i t h a Z e i s s PM-2  linked to a photomultiplier-analog digital  computer-microprocessor  unit.  Each d e t e r m i n a t i o n f o r one nucleus was  r e c o r d e d as t h e mean o f 32 a u t o m a t i c  readings.  A l l q u a n t i f i c a t i o n s were  made u s i n g a 40X (n.a. = 0.75) water-immersion l e n s and a 100 W mercury vapor l i g h t s o u r c e .  Green i n c i d e n t l i g h t was used and, by b a r r i e r  f i l t e r s , t h e e m i t t e d l i g h t measured a t 546 nm. v a l u e s were determined reference.  Twenty-five  R e l a t i v e and a b s o l u t e DNA  on t h e b a s i s o f c o - s t a i n e d c h i c k e n b l o o d as a f i s h e r y t h r o c y t e s and 25 c h i c k e n b l o o d c e l l s were  e v a l u a t e d f o r each s l i d e and two s l i d e s were e v a l u a t e d p e r i n d i v i d u a l .  22 Fluorometric-DNA  1  technique  E r y t h r o c y t e d e n s i t y was v e i n u s i n g a Thomas p i p e t .  determined on b l o o d drawn from t h e The  b l o o d was  caudal  d i l u t e d w i t h Hayem c e l l  c o u n t i n g s o l u t i o n and e r y t h r o c y t e s counted w i t h a Neubauer-ruled hemocytometer. hematocrit  A d d i t i o n a l b l o o d was  obtained with a heparinized  micro-  tube ( S c i e n t i f i c P r o d u c t s , B4416-2) and i t s volume measured.  The  blood was  d i l u t e d t o 1.0 ml w i t h 0.5%  h e p a r i n i n 0.8%  and  kept under r e f r i g e r a t i o n f o r no more than 1 h b e f o r e  sodium c h l o r i d e subsequent  processing. One-hundred and DNA  200  1 known c e l l - d e n s i t y samples were assayed f o r  amount, u s i n g a f l u o r o m e t r i c q u a n t i f i c a t i o n o f a c i d - i n s o l u b l e  d e o x y p u r i n e n u c l e o t i d e s based on the methods of Hinegardner (1971) and F o s t e r and y e a s t RNA  Gurney (1976).  Samples were mixed w i t h 100  as a c a r r i e r and p r e c i p i t a t e d i n 5 ml o f 10%  a c i d f o r 30 min  at 0°C.  cm Whatman GF/C  glass f i b e r f i l t e r s .  HC1  a t 0°C,  dried.  The p r e c i p i t a t e was  2.4  F i l t e r s were r i n s e d t w i c e w i t h 1 M  once w i t h 95% e t h y l a l c o h o l at room t e m p e r a t u r e , and a i r  Each d r i e d f i l t e r was  then e x t r a c t e d f o r 10-15  temperature.  trichloroacetic  c o l l e c t e d by s u c t i o n on  placed i n a v i a l  c o n t a i n i n g 0.25  3,5-diaminobenzoic a c i d d i h y d r o c h l o r i d e f o r 40 min was  g of p u r i f i e d  min w i t h 3 - 5 ml of 1 M HC1  The f l u o r e s c e n c e of t h e HC1  w i t h e x c i t a t i o n at 410 nm.  at 60°C.  The  e x t r a c t was  ml  The  2 M filter  at room  checked at 510  nm  f l u o r e s c e n c e v a l u e s were c a l i b r a t e d  a g a i n s t a c o n t r o l c o n s i s t i n g of a known amount o f p u r i f i e d salmon sperm DNA  (Sigma).  23  RESULTS  Habitat  classification  A l o n g t h e P a c i f i c c o a s t of t h e Olympic P e n i n s u l a , _N. hubbsi were found i n c l o s e d systems, s t a b i l i z e d dune swamps o r bogs ( T a b l e 3 ) .  Such  h a b i t a t s o r i g i n a t e as s t a b i l i z e d beach sediments w i t h water l e v e l s c o n t r o l l e d by r a i n f a l l  and groundwater p e r c o l a t i o n ( H u t c h i n s o n , 1957).  C l o s e d sampling l o c a l i t i e s were r e l a t i v e l y s m a l l , t h e s u r f a c e areas o f both t h e Taholah  and Oyhut s i t e s having been about 0.1 ha ( h e c t a r e s ) .  S i n c e c l o s e d h a b i t a t s do not i n t e r c o n n e c t by means o f waterways, t h e t r a n s f e r o f i n d i v i d u a l s between p o p u l a t i o n s would appear r e s t r i c t e d t o episodes o f f l o o d i n g . The  r e m a i n i n g c o l l e c t i n g s i t e s were i n open systems, r e g i o n s f l o o d e d  by stream o v e r f l o w s  (Table 3 ) .  These h a b i t a t s were l a r g e r than t h e  c l o s e d ones, 2.0 - 6.5 ha, as w e l l as connected s u i t a b l e f o r JJ. h u b b s i .  by streams t o o t h e r s i t e s  _N. hubbsi p r o b a b l y move more r e a d i l y between  p o p u l a t i o n s because o f such i n t e r c o n n e c t i n g waterways and, i n f a c t , _N. hubbsi were i n f r e q u e n t l y caught i n t h e streams f l o w i n g i n t o o r out o f open systems.  C l o s e d systems o c c u r r e d e x c l u s i v e l y i n t h e lowlands  t h e P a c i f i c c o a s t w h i l e open ones were found throughout  along  t h e sampling  area.  Presence  o f f i s h e s o t h e r than N. hubbsi  Attempts were made t o c a p t u r e f i s h e s on at l e a s t f i v e o c c a s i o n s a t t h e l o c a t i o n s l i s t e d i n Table 3.  The Oyhut, Humptulips,  Wishkah,  24  T a b l e 3. S e l e c t e d parameters o f _N. hubbsi c o l l e c t i n g s i t e s . 'Location' i n d i c a t e s t h e c o l l e c t i n g s i t e s used i n t h i s s t u d y , shown i n F i g . I and d e s c r i b e d i n Table 1. S u r f a c e area i s c o n s i d e r e d r e p r e s e n t a t i v e o f h a b i t a t s i z e , s i n c e h a b i t a t s were u s u a l l y s h a l l o w r e l a t i v e l y f l a t d e p r e s s i o n s up t o 1.5 m deep. JN. hubbsi may o c c u r anywhere w i t h i n such waters ( M e l d r i m , 1968). 'Closed' d r a i n a g e s a r e h a b i t a t s m a i n t a i n e d by p r e c i p i t a t i o n , groundwater p e r c o l a t i o n , and r u n o f f . 'Open' d r a i n a g e s are s u s t a i n e d by streams. F i s h e s o t h e r than G. a c u l e a t u s c o - o c c u r r e d w i t h H. hubbsi o n l y i n open d r a i n a g e s i t u a t i o n s .  Location  Surface area (hectares)  Site d r a i nage  Fishes cooccurring w i t h N. h u b b s i *  Tahol ah  0.1  closed  6  Oyhut  0.1  closed  6  Humptul i p s  6.5  open  2, 3, 4, 6, 8  Wi shkah  5.0  open  2, 6, 8  Wynoochee  4.5  open  2, 3, 4, 6, 8  Black River  5.8  open  2, 3, 4, 6, 7, 8  Hanaford  2.0  open  5, 6, 8  Satsop  6.0  open  1, 2, 6, 8  4.5  open  6, 8  Junction  City  *1 = Lampetra s p . ; 2 = Oncorhynchus k i s u t c h Walbaum; 3 = Sal mo gai r d n e r i R i c h a r d s o n ; 4 = R h i n i c h t h y s o s c u l u s "(Gi r a r d ) ; 5 = R i c h a r d s o n i u s b a l t e a t u s (Richardson); 6 = Gasterosteus aculeatus Linnaeus; 7 = Cottus gulosus ( G i r a r d ) ; 8 = C_. asp'er R i c h a r d s o n .  Wynoochee, and Satsop s i t e s were sampled once a month f o r 12 months. Only _N. hubbsi and G a s t e r o s t e u s a c u l e a t u s Linnaeus were caught at c l o s e d sites.  The s t r o n g c o - o c c u r r e n c e o f t h e s e f i s h e s has been d e t a i l e d  McPhail  (1967, 1969)  as w e l l as Hagen e t a l . (1979).  by  G. a c u l e a t u s males  u s u a l l y had b l a c k , i n s t e a d of t h e usual r e d , spawning c o l o r s w i t h i n h a b i t a t s a l s o o c c u p i e d by _N. h u b b s i ; McPhail  (1969) and Hagen e t a l .  (1980) have p r e s e n t e d e x p l a n a t i o n s of t h i s spawning c o l o r polymorphism based on e x p e r i m e n t a l and o b s e r v a t i o n a l d a t a . Several f i s h e s were c a p t u r e d i n open h a b i t a t s .  Red G. a c u l e a t u s  males were r e c o r d e d at t h e J u n c t i o n C i t y s i t e , c o n t r a s t i n g o n l y b l a c k morphs e l s e w h e r e .  Perhaps i m p o r t a n t f i s h e s t o _N. hubbsi were t h e  s a l m o n i d s , Onchorynchus k i s u t c h (Walbaum) and Sal mo g a i r d n e r i as w e l l  as two c o t t i d s , Cottus gulosus  ( G i r a r d ) and C_. a s p e r  Richardson, Richardson,  s i n c e t h e s e s p e c i e s are p i s c i v o r o u s (Moodie and Reimchen, 1976). A l t h o u g h salmonids were not recorded from a l l l o c a l i t i e s t h e s e c o u l d have been p r e s e n t i f c o t t i d s were c a p t u r e d . documented a s t r o n g c o - o c c u r r e n c e  Moodie and Reimchen (1976) have  of t h e s e t a x a .  The n o n - p a r a s i t i c Lampetra sp. c a p t u r e d at t h e Satsop s i t e had a unique d e n t i t i o n f i t t i n g no a v a i l a b l e k e y s , p r o b a b l y an example o f t h e endemism w i t h i n t h e C h e h a l i s R i v e r system noted by McPhail c y p r i n i d i n open systems was  (1967).  most-often  encountered  (Girard).  S i n c e R. o s c u l u s i s p r i m a r i l y i n s e c t i v o r o u s ( S c o t t and  The  Rhinichthys osculus  Crossman, 1972), i t i s l i k e l y not a s i g n i f i c a n t p r e d a t o r on _N. h u b b s i . P h y s i c a l and chemical d e t e r m i n a t i o n s on t h e  water  Waters at a l l c o l l e c t i n g s i t e s but one c o n s i s t e n t l y had a c i d i c 4.5-6.5, and no d e t e c t a b l e d i s s o l v e d s a l t s .  The J u n c t i o n C i t y s i t e  pH's, was  26 an e x c e p t i o n a l  h a b i t a t f o r _N. h u b b s i , s i n c e monthly d e t e r m i n a t i o n s from  February - June 1979 y i e l d e d pH's between 7.2 and 7.4 as w e l l as 2-4 ppt ( p a r t s per thousand) d i s s o l v e d  salts.  The l o w e s t pH's, 4.5 - 5.0, were r e c o r d e d d u r i n g t h e 23-29 J u l y 1979 f i e l d t r i p s t o t h e Oyhut and Satsop s i t e s , when r a i n f a l l a p p r o a c h i n g t h e y e a r l y minimum ( R u f f n e r , 1980).  had been  E v a p o r a t i o n had reduced  t h e Oyhut and Satsop c o l l e c t i n g s i t e s from l a r g e r c o n t i n u o u s bodies o f water t o s e r i e s o f s m a l l e r p o o l s .  No oxygen was d e t e c t e d i n some o f t h e  l a t t e r a t Satsop, i n c o n t r a s t t o 4-7 p a r t s per m i l l i o n e l s e w h e r e .  Carbon  d i o x i d e c o n t e n t s i n Oyhut and Satsop waters d u r i n g t h i s p e r i o d o f t i m e were as much as 5-6 p p t , l i k e l y c o n t r i b u t i n g t o t h e low pH's. temperatures everywhere  Water  had ranged from 4°C i n t h e w i n t e r t o 23°C d u r i n g  t h e summer, but some p o o l s a t t h e Satsop s i t e were 27-30°C on 23 J u l y 1979.  Meristic  characteristics  _N. hubbsi from t h e C o p a l i s R i v e r and Conner Creek d r a i n a g e s had been c a p t u r e d i n 1947 and 1968, r e s p e c t i v e l y .  These specimens  from t h e U n i v e r s i t y o f Washington c o l l e c t i o n .  were  borrowed  T h e i r p r e s e r v a t i o n was  such t h a t fewer m e r i s t i c c h a r a c t e r s were e v a l u a t e d than on specimens caught by m y s e l f . obtained.  Counts o f t h e median and p a i r e d f i n rays were  S c a l e c o u n t s , w i t h t h e e x c e p t i o n o f s c a l e s above t h e l a t e r a l  s c a l e row, were not done because t o o many f i s h were misshapen missing scales. fish;  Lateral  and/or  bar counts on males were t a k e n from some C o p a l i s  however, t h e r e m a i n i n g U n i v e r s i t y o f Washington specimens  faded o r d i s c o l o r e d f o r a c c u r a t e e v a l u a t i o n o f t h i s c h a r a c t e r .  were t o o Small  numbers o f N. hubbsi were o b t a i n e d from t h e B l a c k R i v e r and Hanaford  27 Creek c o l l e c t i n g s i t e s , so m e r i s t i c data from t h e s e f i s h were not included i n the analyses. There was no s t r o n g c o r r e l a t i o n between m e r i s t i c c h a r a c t e r s t a t e s and s t a n d a r d l e n g t h , a measure o f s i z e (Table 2 ) . S i m i l a r l y ,  Chernoff  (1982) found t h a t m e r i s t i c c h a r a c t e r s on a t h e r i n i d s were not c o r r e l a t e d w i t h mensural ones.  Only a d u l t _N. hubbsi were e v a l u a t e d , r a n g i n g from  3.43 - 6.68 cm SL f o r females  and 3.19 - 6.19 cm SL f o r m a l e s , so t h e  absence o f j u v e n i l e specimens c o u l d have been p a r t l y r e s p o n s i b l e f o r t h e lack of c o r r e l a t i o n .  S i n c e s i z e d i d not appear t o have i n f l u e n c e d  m e r i s t i c c h a r a c t e r s t a t e s , t h e l a t t e r were a n a l y z e d by means o f univariate statistical  Sexual  t e s t s instead of analysis of covariance.  dimorphism o f d o r s a l and anal f i n rays  The d i s t r i b u t i o n s o f d o r s a l and anal f i n r a y counts  appeared  s e x u a l l y - d i m o r p h i c w i t h i n some p o p u l a t i o n s (Table 4 ) . When samples from any c o l l e c t i n g s i t e were s o r t e d by s e x , males u s u a l l y had h i g h e r than d i d females  counts  (Tables 5-8). Both sexes were found t o have t h e same  modal d o r s a l f i n c o u n t , 13 r a y s , a t a l l s i t e s but two.  Tahol ah and Oyhut  males had modal d o r s a l f i n counts o f 14 rays (Table 6) c o n t r a s t e d by o n l y 13 rays f o r females  (Table 7 ) .  Wishkah and Humptulips males had modal counts o f 11 anal f i n r a y s , i n c o n t r a s t t o males from elsewhere which had 12 (Table 8 ) . A s i m i l a r d i s t r i b u t i o n o f modal anal f i n r a y counts was found i n f e m a l e s ,  although  Tahol ah specimens had a mode o f o n l y 11 rays and t h o s e from Conner Creek were b i m o d a l , w i t h 11 and 12 rays (Table 9 ) .  Dorsal f i n rays i n males ( T a b l e s 6, 10; F i g u r e I I ) Mean d o r s a l f i n r a y counts were s e p a r a b l e i n t o two c a t e g o r i e s .  The  2 8  T a b l e 4. C h i - s q u a r e a n a l y s e s on t h e d i s t r i b u t i o n s o f d o r s a l and anal f i n ray numbers compared between sexes by l o c a t i o n f o r N. h u b b s i . Although s e v e r a l s c a l e and f i n ray c h a r a c t e r s were examined Tn t h i s s t u d y , o n l y d o r s a l and anal f i n rays were found t o be s e x u a l l y - d i m o r p h i c , (n.s. = not s i g n i f i c a n t ) . Dorsal  f i n rays:  Location  Chi-square  d.f.  P.  Tahol ah  2.799  1  n.s.  Copali s  1.714  1  n.s.  Conner Creek  3.776  2  n.s.  12.228  2  X 0.005(2)=10.597, reject null  0.222  1  n.s.  42.243  2  X 0.001(2)=13.16, reject null  Wynoochee  3.418  3  n.s.  Satsop  4.159  2  n.s.  Oyhut Humptuli ps Wi shkah  Anal  2  2  f i n rays:  Location  Chi-square  d.f.  Tahol ah Copal i s  2.334  1  n.s.  Conner Creek  0.948  1  n.s.  Oyhut  1.437  2  n.s.  Humptul i ps  2.449  1  n.s.  Wi shkah  0.187  1  n.s.  Wynoochee  0.250  1  n.s.  14.707  3  X 0.005(3)=12.838, reject null  Satsop  2  T a b l e 5. Sexual dimorphism i n t h e mean number of d o r s a l and anal f i n rays i n J N . hubbsi e v a l u a t e d w i t h t - t e s t s . A l l comparisons between sexes a r e s i g n i f i c a n t a t t h e p=0.001 l e v e l . Males had g r e a t e r numbers of rays on average t h a n d i d f e m a l e s . Dorsal f i n ray  Anal f i n ray  counts  counts  t-test  df  significance  t0.001(2)=4.02  32.37  17  t0.001(2)=3.97  38  t0.001(2)=3.57  10.68  38  t0.001(2)=3.57  37.03  51  t0.001(2)=3.50  49.16  51  t0.001(2)=3.49  125.53  65  t0.001(2)=3.45  23.92  64  t 0 . 0 0 1 ( 2 ) = 3.45  Humptulips  29.83  32  t0.001(2)=3.62  21.79  32  t0.001(2)=3.62  Wishkah  75.73  81  t0.001(2)=3.41  24.33  80  t 0 . 0 0 1 ( 2 ) = 3.42  Wynoochee  12.47  38  t0.001(2)=3.57  33.93  38  t0.001(2)=3.57  Satsop  73.32  67  t0.001(2)=3.43  49.67  66  t0.001(2)=3.44  t-test  df  significance  Tahol ah  18.38  17  Copal i s  32.59  Conner Creek  Location  Oyhut  Table 6. Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r t h e number o f d o r s a l f i n rays i n male_N. hubbsi. 'R' i s t h e o r d e r i n g o f l o g a r i t h m i c means a c c o r d i n g t o i n c r e a s i n g magnitude and i d e n t i f i e s t h e p o p u l a t i o n s as used i n t h e Student-Newman-Keuls t e s t (R, T a b l e 10b). Number of d o r s a l f i n rays  Location  11  12  Tahol ah  13  14  4  5  15  1  Logarithm!' c mean  s.d.  Arithmetic mean  95% c o n f i dence i nterval  R_  9  1.1318  0.0431  13.56  12.55  -  15.62  7  28  1.1253  0.0206  13.36  13.10  -  13.59  6  26  1.1249  0.0214  13.35  13.06  -  13.60  5  33  1.1369  0.0244  13.76  13.43  -  13.98  8  Copal i s  1  17  9  Conner Creek  2  13  11  Oyhut  1  12  15  Humptuli ps  3  11  2  16  1.1114  0.0200  12.94  12.61  -  13.24  1  Wishkah  7  32  9  48  1.1149  0.0200  13.04  12.86  -  13.20  4  2  12  6  22  1.1130  0.0305  13.00  12.57  -  13.38  2  4  24  5  33  1.1146  0.0184  13.03  12.82  13.21  3  Wynoochee Satsop  2  1  5  Table 7. Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r t h e number o f d o r s a l f i n rays i n female N. hubbsi. Number o f d o r s a l f i n rays  s.d.  95% confidence i nterval  13  14  1  6  3  10  1.1201  0.0210  13.20  12.74  - 13.65  9  3  12  1.1220  0.0147  13.25  12.96  - 13.53  1  18  6  27  1.1194  0.0247  13.19  12.87  - 13.46  Oyhut  3  20  11  34  1.1213  0.0201  13.24  13.01  - 13.44  Humptuli ps  3  15  18  1.1081  0.0136  12.83  12.72  - 13.03  Wishkah  5  28  1  34  1.1098  0.0143  12.88  12.73  - 13.02  1  13  3  18  1.1198  0.0378  13.17  13.00  - 13.36  3  21  11  35  1.1211  0.0198  13.23  13.01  - 13.45  11  Tahol ah Copal i s Conner Creek  Wynoochee Satsop  1  1  1  N  Arithmeti c mean  12  Location  15  Logarithmi c mean  T a b l e 8. Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r t h e number o f anal f i n rays i n male JN. hubbsi. 'R' i s t h e o r d e r i n g o f l o g a r i t h m i c means a c c o r d i n g t o i n c r e a s i n g magnitude and i d e n t i f i e s t h e p o p u l a t i o n s as used i n t h e Student-Newman-Keuls t e s t (R, T a b l e 13b). Number o f anal f i n rays  Location  11  11  12  13  _N  Logarithmi c mean  s.d.  Arithmetic mean  confidence i nterval  R  Tahol ah  1  2  6  9  1.0620  0.0282  11.56  10.98 - 12.11  3  Copalis  2  8  18  28  1.0627  0.0246  11.57  10.98 - 11.42  5  11  13  2  26  1.0659  0.0233  11.65  11.39 - 11.89  6  1  32  1.0624  0.0235  11.56  11.32 - 11.77  4  Conner Creek Oyhut  1  13  17  Humptuli ps  1  10  5  16  1.0506  0.0223  11.25  10.93 - 11.55  1  32  16  48  1.0540  0.0181  11.33  11.19 - 11.46  2  6  15  1  22  1.0705  0.0196  11.77  11.53 - 11.99  7  3  24  4  33  1.0752  0.0257  11.91  11.64 - 12.14  8  Wishkah Wynoochee Satsop  2  CJO  ro  Table 9. Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r t h e number o f anal f i n rays i n female _N. hubbsi. 'R' i s t h e o r d e r i n g o f l o g a r i t h m i c means a c c o r d i n g t o i n c r e a s i n g magnitude and i d e n t i f i e s t h e p o p u l a t i o n s as used i n t h e Student-Newman-Keuls t e s t (R, Table 14b). Number o f anal f i n rays  Location  10  Tahol ah Copalis  1  Conner Creek  13  1  Logarithmic mean  s.d.  Arithmetic mean  95% confidence i nterval  _11  _12  7  3  10  1.0527  0.0182  11.30  10.96  - 11.63  3  8  3  12  1.0474  0.0225  11.17  10.79  - 11.53  1  R_  14  13  27  1.0596  0.0192  11.48  11.27  - 11.67  6  12  19  34  1.0589  0.0258  11.47  11.22  - 11.69  5  Humptuli ps  15  3  18  1.0477  0.0145  11.17  10.98  - 11.34  2  Wishkah  21  13  34  1.0558  0.0186  11.38  11.20  -  U.54  4  6  12  18  1.0666  0.0183  11.67  11.41  - 11.90  7  9  24  35  1.0682  0.0218  11.71  11.50  - 11.90  8  Oyhut  3  Wynoochee Satsop  1  1  33 34  Table 10. S t a t i s t i c a l a n a l y s e s u s i n g l o g a r i t h m i c means t e s t i n g f o r i n t e r p o p u l a t i o n a l d i f f e r e n c e s i n t h e number o f d o r s a l f i n rays i n male N. hubbsi: (a) a n a l y s i s o f v a r i a n c e and (b) Student-Newman-Keuls (SNK) f o r paTrwise comparisons. 'R' i n t h e SNK i d e n t i f i e s t h e p o p u l a t i o n means i n Table 6; s i g n i f i c a n c e l e v e l s a r e denoted by s u p e r s c r i p t s t a r s ( 0.05=*; 0.01=**; 0.001=***). a.  A n a l y s i s of v a r i a n c e f o r data i n Table 6 Source of V a r i a t i o n  Sums o f Squares  df  Mean Squares  Total  0.1128  214  Groups  0.0163  7  0.0023  Error  0.0965  207  0.0005  F = 4.60 F 0.0005(1) 7,207 = 3.92; r e j e c t n u l l Levene's t e s t f o r equal v a r i a n c e s = 2.94; p < 0.02 One-way a n a l y s i s of v a r i a n c e , t e s t s t a t i s t i c s f o r w i t h i n - g r o u p v a r i a n c e s not assumed t o be e q u a l : Welch = 4.60; p < 0.005 Brown-Forsythe = 4.65; p < 0.005 b.  Student .-Newman-Keuls t e s t f o r R  R  X  2  1  X  1.1114  1.1130  N  16  22  data i n  3  Table 6 4  1.1146  6  5  7  1.1149  1.1249  1.1253  1.1318  1.1369  48  26  28  9  33  34  N  -  1  1.1114  16  2  1.1130  22  0.0016  3  1.1146  34  0.0032  0.0016  4  1.1149  48  0.0035  0.0019  0.003  5  1.1249  26  0.0135  0.0119  0.0103  0.0100  6  1.1253  28  0.0139  0.0123  0.0107  0.0104  0.0004  7  1.1318  9  0.0204  0.0188  0.0172  0.0169  0.0069  0.0065  8  1.1369  33  0.5220 0.0120  0.0116  -  -  0.5255 0.5239 0.5223  8  -  -  -  0.0051  15  > co ^  14  e  To 2 w E  o  r  28 22  33  0  13  Q  12  I m o SI CO  CO  a o O  I  I  a>  <u O c c o  o  I  I  a  o  a  E I  I CD  in  CO  o o o c >>  a o  m +~  CO  CO  5  F i g u r e I I . Geographic v a r i a t i o n i n t h e number of d o r s a l f i n rays i n male_N. h u b b s i . A r i t h m e t i c mean i s r e p r e s e n t e d as t h e c e n t e r o f t h e c r o s s - h a t c h e d c i r c l e , upper and l o w e r 9 5 % c o n f i d e n c e i n t e r v a l s as t h e v e r t i c a l l i n e s . The number immediately above t h e upper c o n f i d e n c e i n t e r v a l i s sample s i z e .  36. H u m p t u l i p s , Wishkah, Wynoochee, and Satsop c o l l e c t i o n s were e q u a l , w i t h a combined mean o f 13.02 r a y s .  statistically  The T a h o l a h , Oyhut, Copal i s  and Conner Creek samples were a l s o i n d i s t i n g u i s h a b l e from each o t h e r , w i t h a combined mean o f 13.51 r a y s .  SMC i n d i c a t e d a s t a t i s t i c a l  d i f f e r e n c e between t h e s e two groupings ( T a b l e 1 1 ) . assemblage  The more western  ( T a h o l a h , Copal i s , Conner Creek and Oyhut) had a g r e a t e r  combined mean d o r s a l f i n r a y count than d i d t h e e a s t e r n one ( H u m p t u l i p s , Wishkah, Wynoochee and S a t s o p ) . Dorsal f i n rays i n females ( T a b l e s 7, 12; F i g u r e I I I ) ANOVA i n d i c a t e d no s i g n i f i c a n t d i f f e r e n c e s between p o p u l a t i o n s r e g a r d i n g d o r s a l f i n ray counts i n f e m a l e s . Anal f i n rays i n males ( T a b l e s 8, 13; F i g u r e IV) There were no s i g n i f i c a n t d i f f e r e n c e s between samples from s i t e s west o f and i n c l u d i n g Wishkah. statistically  Wynoochee and Satsop means counts were  equal t o each o t h e r , but counts from both d i f f e r e d from t h e  Wishkah mean a t t h e p = 0.05 and p = 0.001 l e v e l s r e s p e c t i v e l y .  The  Humptulips sample mean d i f f e r e d from t h e Satsop one a t t h e p = 0.01 level.  SMC i n d i c a t e d a s i g n i f i c a n t d i f f e r e n c e between combined Wynoochee  and Satsop mean counts compared a g a i n s t combined Wishkah and Humptulips ones, n o n - s i g n i f i c a n c e f o r Humptulips and Wishkah mean counts compared a g a i n s t t h o s e from o t h e r samples from west o f t h e Montesano H i l l s 11).  The c o a s t a l  (Table  p o p u l a t i o n s appeared t o have been equal r e g a r d i n g mean  anal f i n r a y counts  Average counts may have been l e s s a t Humptulips and  Wishkah, but w i t h i n - p o p u l a t i o n sample s i z e s were t o o s m a l l , p r o h i b i t i n g statistical  v e r i f i c a t i o n of t h i s p o s s i b i l i t y .  Mean counts s i g n i f i c a n t l y  i n c r e a s e d from 11.33 rays a t Wishkah t o 11.77 rays a t a t Wynoochee.  37  T a b l e 11. S c h e f f e ' s m u l t i p l e c o n t r a s t s t e s t s on t h e mean d o r s a l and anal f i n r a y counts i n males and anal f i n r a y counts i n females f o r N. hubbsi. 'X' r e f e r s t o p o p u l a t i i o n mean, w i t h each p o p u l a t i o n i d e n t i f i e d "by a s u b s c r i p t number (R i n Table 6 f o r d o r s a l f i n rays i n m a l e s , Table 8 f o r anal f i n rays i n males, Table 9 f o r anal f i n rays i n f e m a l e s ) . See t e x t f o r an e x p l a n a t i o n o f r e s u l t s .  Standard error  Contrast Dorsal  ray means , male  x +x +x +x 5  6  Conclus i o n  7  x +x +x +x  8  1  2  _  3  4  _  0.0010  16.20  = 0.0162 Anal  r a y means, male  X +X 7  SO.0005(1) 7,206=5.24; p<0.0005; r e j e c t n u l l  XJ+X2+X3+X4+X5+X5  8  2 0.0132  x x 7+  Xj + X  8  2 = 0.0205  Xj+X  2  Anal  3.47  SO.05(1) 7,208=3.79; p>0.05, accept n u l l  0.0045  4.56  SO.01(1),7,208 = 4.37; p<0.0005; r e j e c t n u l l  0.0042  2.62  SO.05(1) 7,208=3.79; p>0.05; accept n u l l  0.0033  1.48  SO.05(1) 7,180-3.80; p>0.05,accept n u l l  2  2  x +x x x x 3  2 = 0.0110  0.0038  4  4  5  6  4  r a y means, female  Xj+X^+Xs+Xy _  = 0.0049  x +x +x +x 3  4  4  6  8  38  Table 12. A n a l y s i s o f v a r i a n c e on mean d o r s a l f i n r a y counts i n female i i * hubbsi c a p t u r e d a t s e v e r a l l o c a l i t i e s . The d a t a i s p r e s e n t e d i n Table 7. No d i f f e r e n c e s between p o p u l a t i o n samples were i n d i c a t e d .  Source of V a r i a t i o n  Sums o f Squares  Total  0.0880  187  Groups  0.0052  7  0.0007  Error  0.0828  180  0.0005  df  Mean Squares  F = 1.40 F 0.05(1), 7,180 = 2.06; accept n u l l S i n c e no d i f f e r e n c e s were i n d i c a t e d , t h e d a t a was not f u r t h e r a n a l y z e d w i t h a Student-Neuman-Keuls t e s t .  14 10  CO CO  34  18  35  Q)  18  — «j co E 13  34  CO <D O w  o  12  I « o co  CO  a  o O  I  I  I  CD  D .C  a  0)  k— O 0)  c c o O  o  a E X  co (0  CD  o o o c >>  a o m  ca CO  5  Figure III. Geographic v a r i a t i o n i n the number of d o r s a l f i n rays i n female N. an e x p l a n a t i o n of symbols.  hubbsi.  See F i g . II  for -^o  40  T a b l e 13. S t a t i s t i c a l a n a l y s e s u s i n g l o g a r i t h m i c means t e s t i n g f o r i n t e r p o p u l a t i o n a l d i f f e r e n c e s i n t h e number o f anal f i n rays i n male N. hubbsi: (a) a n a l y s i s o f v a r i a n c e and (b) Student-Newman-Keuls (SNK) f o r p a i r w i s e comparisons. 'R' i n t h e SNK i d e n t i f i e s t h e p o p u l a t i o n means i n Table 8; s i g n i f i c a n c e l e v e l s a r e denoted by s u p e r s c r i p t a s t e r i s k s ( 0.05=* ; 0.01=**; 0.001=***).  a.  Analysis of variance  f o r data i n Table 8  Source of V a r i a t i o n  Sums o f Squares  df  Mean Squares  Total  0.1178  213  Groups  0.0127  7  0.0018  Error  0.1051  206  0.0005  F = 3.60 F 0.0025(1) 7,206 = 3.29; r e j e c t n u l l Levene's t e s t f o r equal v a r i a n c e s = 2.97; p < 0.02 One-way a n a l y s i s o f v a r i a n c e , t e s t s t a t i s t i c s f o r w i t h i n - g r o u p v a r i a n c e s not assumed t o be e q u a l : Welch = 3.00; p < 0.005 Brown-Forsythe = 3.16; p < 0.005 b.  Student-Newman-Keuls t e s t f o r d a t a i n T a b l e 8 R  1  2  X  1.0506  1.0540  N  16  48  3  4 1.0620  5  6  7  8  1.0624  1.0627  1.0659  1.0705  1.0752  32  28  26  22  33  9  R  X  1  1.0506  16  2  1.0540  48  0.0034  3  1.0620  9  0.0114  0.0080  4  1.0624  32  0.0118  0.0084  0.0004  5  1.0627  28  0.0121  0.0087  0.0007  0.0003  6  1.0659  26  0.0153  0.0119  0.0039  0.0035  0.0032  7  1.0705  22  0.0199  0.0081  0.0078  0.0046  8  1.0752  33  0.0165 0.0085 0.Q&U 0.6M2 0.0132  0.0128  0.0125  0.0093  N  -  0.0047  ro - n X -"• — o> 3 cu r+ -'• O 3 1  £Z - J  rt>  i—i < •  O !7> - h ro O I/)  << 3 cr O —J  •  cQ  -5 Cu -o 3" -J.  o  <  r  Anal rays (male)  T  T"  Taholah  3  Copalis  r-t3" ro  vX  ">  3  cr ro  cu 3 Cu  Conner  /TV l^l  Creek  Oyhut  —  /TV  K  0  O)  Humptulips cu  Wishkah 3  CU  cr  ro ro  o cu  ///  Wynoochee  —  Satsop  —  /TV  I  42* Anal f i n rays i n females ( T a b l e s 9, 14; F i g u r e V) The o n l y p a i r - w i s e comparisons i n w h i c h s t a t i s t i c a l l y  significant  d i f f e r e n c e s were found i n c l u d e d Satsop f i s h compared w i t h C o p a l i s specimens  and Satsop f i s h w i t h Humptulips ones, t h e Satsop c o l l e c t i o n s  h a v i n g t h e l e s s e r mean i n both c a s e s .  SMC d i d not i n d i c a t e o t h e r  i n t e r s a m p l e d i f f e r e n c e s ( T a b l e 1 1 ) . Geographic t r e n d s i n c h a r a c t e r v a r i a t i o n were not obvious i n SNK, but t h o s e p o p u l a t i o n s w i t h l e s s e r mean counts i n an a b s o l u t e sense o c c u r r e d west o f t h e Wynoochee c o l l e c t i n g site.  C o p a l i s and Conner Creek specimens were c a p t u r e d 32 and 11 y e a r s ,  r e s p e c t i v e l y , b e f o r e t h e o t h e r samples, which may have been a f a c t o r i n p r o d u c i n g mean counts s i g n i f i c a n t l y d i f f e r e n t from t h a t o b t a i n e d from Satsop  fish.  Caudal f i n rays (Tables 15, 16; F i g u r e V I ) Caudal f i n r a y means were s t a t i s t i c a l l y - i n d i s t i n g u i s h a b l e between T a h o l a h , Oyhut, H u m p t u l i p s , Wishkah, Wynoochee, and Satsop  samples.  C o p a l i s and Conner Creek mean counts were g r e a t e r than t h e Wynoochee and Satsop ones a t t h e p = 0.05 l e v e l .  No t r e n d s i n c h a r a c t e r v a r i a t i o n were  suggested by t h e a n a l y s e s e x c e p t f o r d i f f e r e n c e s r e s u l t i n g from t h e t i m e when C o p a l i s and Conner Creek specimens were c o l l e c t e d . P e c t o r a l f i n r a y s ( T a b l e s 17, 18; F i g u r e V I I ) The SNK i n d i c a t e d t h a t n e a r e s t - n e i g h b o r sample means were not d i f f e r e n t from each o t h e r , but t h a t t h e r e was a c l i n a l ray  counts from west t o e a s t .  i n c r e a s e i n mean  T a h o l a h , C o p a l i s , Conner Creek, and Oyhut  mean counts appeared e q u a l , t h e i r combined mean having been 19.63 pectoral f i n rays.  The next e q u i v a l e n t group i n c l u d e d Oyhut, H u m p t u l i p s ,  Wishkah and Wynoochee specimens w i t h a combined mean o f 20.20 r a y s .  The  43  Table 14. S t a t i s t i c a l a n a l y s e s u s i n g l o g a r i t h m i c means t e s t i n g f o r i n t e r p o p u l a t i o n a l d i f f e r e n c e s i n t h e number o f anal f i n rays i n female N. h u b b s i : (a) a n a l y s i s o f v a r i a n c e and (b) Student-Newman-Keuls (SNK) f o r p a i r w i s e comparisons. 'R' i n t h e SNK i d e n t i f i e s t h e p o p u l a t i o n means i n Table 9; s i g n i f i c a n c e l e v e l s are denoted by s u p e r s c r i p t a s t e r i s k s ( 0.05=*).  a.  A n a l y s i s o f v a r i a n c e f o r d a t a i n Table 9 of  Sums Squares  Mean Squares  Source Variation  of  Total  0.0856  187  Groups  0.0087  7  0.0012  Error  0.0769  180  0.0004  df  F = 3.00 F 0.01(1) 7,180 = 2.74; r e j e c t n u l l Levene's t e s t f o r equal v a r i a n c e s = 1.55; p > 0.10 S i n c e t h e v a r i a n c e appeared homodescacic, Welch and BrownF o r s y t h e t e s t s were n o t performed. b.  Student-Newman-Keuls t e s t f o r d a t a i n Table 9 R  n K  _  X  1  2  X  1.0474  1.0477  N  12  18  4  3 1.0527  5  6  8  7  1.0558  1.0589  1.0596  1.0666  1.0682  34  34  27  18  35  10  M IN  1  1.0474  12  2  1.0477  18  0.0003  3  1.0527  10  0.0053  0.0050  4  1.0558  34  0.0084  0.0081  0.0031  5  1.0589  34  0.0115  0.0112  0.0062  0.0031  6  1.0596  27  0.0122  0.0119  0.0069  0.0038  0.0007  7  1.0666  18  0.0192  0.0189  0.0139  0.0108  0.0077  0.0076  8  1.0682  35  0.0208  0.02*05  0.0155  0.0124  0.0093  0.0086  _  -  0.0016  -  12  18  '12  27  34  35  34 18  O  gi 10«-  _C0  Tar  o  I </) To a Co  I  I  I  I co  CD CD  O CD  C c  -C  O>.  a a E 3  I  ca  CD CD  JO  o o o c >.  I a o  co ca 03  o O  F i g u r e V. Geographic v a r i a t i o n i n the number of anal f i n rays i n female N. h u b b s i . e x p l a n a t i o n of symbols.  See F i g . I I f o r an  Table 15. Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r t h e number o f caudal f i n rays i n N. h u b b s i . 'R' i s t h e o r d e r i n g o f l o g a r i t h m i c means a c c o r d i n g t o i n c r e a s i n g magnitude and i d e n t i f i e s t h e p o p u l a t i o n s as used i n t h e Student-Newman-Keuls t e s t (R, Table 16b).  Number o f caudal f i n rays  Ii  Arithmeti c mean  95% confidence i nterval  _N  Logarithm!' c mean  s.d.  19  1.2527  0.0078  17.89  17.74 -• 18.05  4  i i XL  18  2  17  2  49  3  55  1.2547  0.0101  17.98  17.86 -• 18.09  8  Conner Creek  4  47  2  53  1.2543  0.0082  17.96  17.86 -• 18.05  6  Oyhut  5  59  64  1.2533  0.0067  17.92  17.85 -• 17.99  5  Humptul i p s  2  31  1  34  1.2545  0.0078  17.97  17.86 -• 18.08  7  2  81  1.2491  0.0127  17.75  17.63 -• 17.86  3  Location Tahol ah Copalis  1  R_  Wishkah  1  20  58  Wynoochee  4  5  31  40  1.2471  0.0170  17.68  17.44 •• 17.88  1  Satsop  7  6  55  68  1.2478  0.0165  17.71  17.53 -• 17.86  2  46  T a b l e 16. S t a t i s t i c a l a n a l y s e s u s i n g l o g a r i t h m i c means t e s t i n g f o r i n t e r p o p u l a t i o n a l d i f f e r e n c e s i n t h e number o f caudal f i n rays i n \L hubbsi: (a) a n a l y s i s o f v a r i a n c e and (b) Student-Newman-Keuls t e s t (SNK) f o r p a i r w i s e comparisons. 'R' i n t h e SNK i d e n t i f i e s t h e p o p u l a t i o n means i n Table 15; s i g n i f i c a n c e l e v e l s a r e denoted by s u p e r s c r i p t a s t e r i s k s ( 0.05=*).  a.  A n a l y s i s of v a r i a n c e f o r data i n Table 15 Source of V a r i a t i o n  Sums o f Squares  Mean Squares  df  Total  0.0608  413  Groups  0.0037  7  Error  0.0571  406  0.005 0.0001  F = 5.00 F 0.0005(1) 7,406 = 3.83, r e j e c t n u l l Levene's t e s t f o r equal v a r i a n c e s = 13.41; p < 0.001 One-way a n a l y s i s o f v a r i a n c e , t e s t s t a t i s t i c s f o r w i t h i n - g r o u p v a r i a n c e s not assumed t o be e q u a l : Welch = 3.62; p < 0.005 Brown-Forsythe = 3.15; p < 0.009 b.  Student-Newman-Keuls t e s t f o r data i n Table 15 R  R  X  1  2  3  X  1.2471  1.2478  1.2491  1.2527  1.2533  1.2543  N  40  68  81  19  64  53  4  5  6  7  8  1.2545 1.2547 34  55  N  1 1.2471 40  -  2 1.2478 68  0.0008  3 1.2491 81  0.0020 0.0013  -  4 1.2527 19  0.0056 0.0049  0.0036  -  5 1.2533 64  0.0062 0.0055  0.0042  0.0006  -  6 1.2543 53  0.0072 0.0065  0.0052  0.0016  0.0010  -  7 1.2545 34  0.0074 0.0067  0.0054  0.0018  0.0012  0.0002  -  8 1.2547 55  0.0$76 0.0069  0.0056  0.0020  0.0014  0.0004  0.0002  -  rt) X  -n  —*• CO  T3 —' c cu 3 rt> Ol rt- < O • 3 O  -t! rt> o </>  v<  3  us -J CU  Caudal rays  cr-o o 3in o  N  r  < CU  o 3  rt) 3  3  00  Taholah  —  -(Jhw  Copalis  —  <J>5  Conner Creek  —  cr n> Oyhut  —  $ 2  o CU  Q. CU  Humptulips  "5  Wishkah  CU *<  c cr cr  oo rt>  CU  3  —  —  <J>~!  Wynoochee  —  -<3>-o  Satsop  —  -($)-£  •o  Table 17. Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and g e o m e t r i c means f o r t h e number o f p e c t o r a l f i n rays i n N. h u b b s i . 'R' i s t h e o r d e r i n g o f l o g a r i t h m i c means a c c o r d i n g t o i n c r e a s i n g magnitude and i d e n t i f i e s t h e p o p u l a t i o n s as used i n t h e Student-Newman-Keuls t e s t (R, T a b l e 18b). Number o f p e c t o r a l f i n rays  Location  17 18 19 20 21 22 23  95% confidence i nterval  _N  Logarithmic mean  s.d.  19  1.2785  0.0152  19.00  18.67  - 19.31  1  Arithmetic mean  R_  Tahol ah  5  Copal i s  5 15 20  9  49  1.2934  0.0201  19.67  19.39  - 19.91  3  2 25 19  1  49  1.2854  0.0169  19.31  19.07  - 19.51  2  Oyhut  2  9 24  5  40  1.2964  0.0172  19.80  19.54 - 20.04  4  Humptulips  1  4 13 10  28  1.3038  0.0191  20.14  19.78  - 20.47  5  l ' 22 30  53  1.3126  0.0149  20.55  20.35 - 20.74  7  40  1.3049  0.0211  20.20  19.86  - 10.50  6  68  1.3183  0.0176  20.82  20.61  - 21.02  8  Conner Creek  2  Wishkah  9  5  Wynoochee  2  6 15 16  1  Satsop  1  3  4  9 50  1  CO  49  Table 18. S t a t i s t i c a l a n a l y s e s u s i n g l o g a r i t h m i c means, t e s t i n g f o r i n t e r p o p u l a t i o n a l d i f f e r e n c e s i n t h e number o f p e c t o r a l f i n rays i n NI. hubbsi: (a) a n a l y s i s o f v a r i a n c e and (b) Student-Newman-Keuls (SNK) f o r p a i r w i s e comparisons. 'R' i n t h e SNK i d e n t i f i e s t h e p o p u l a t i o n means i n Table 17; s i g n i f i c a n c e l e v e l s a r e denoted by s u p e r s c r i p t a s t e r i s k s ( 0.005=*; 0.01=**; 0.001=***).  a.  A n a l y s i s of variance  f o r d a t a i n Table 17  Source of V a r i a t i o n  Sums o f Squares  df  Mean Squares  Total  0.5661  345  Groups  0.2299  7  0.0328  Error  0.3362  338  0.0010  F = 32 80 F 0.0005(1) 7,338 = 384; r e j e c t n u l l Levene's t e s t f o r equal v a r i a n c e s = 2.93; p < 0.014 One-way a n a l y s i s o f v a r i a n c e , t e s t s t a t i s t i c s f o r w i t h i n - g r o u p v a r i a n c e s not assumed t o be e q u a l : Welch = 32.68; p < 0.001 Brown-Forsythe = 31.98; p < 0.001 b.  Student-Newman-Keuls t e s t f o r data i n Table 17 R  R  X  2  1  x  1.2785  1.2854  N  19  49  3  4 1.2934  5  6  7  1.2964  1.3038  1.3049  1.3126  40  28  40  53  49  N  -  1  1.2785  19  2  1.2854  49  0.0069  3  1.2934  49  0.0149  0.0080  4  1.2964  40  0.0179  0.0110  0.0030  5  1.3038  28  0.02*53  0.0l84  0.0104  0.0074  6  1.3049  40  0.0264  0.0195  0.0115  0.0085  0.0011  7  1.3126  53  0.6541 oMIz  0.0f92  0.0162  0.0088  0.0077  8  1.3183  68  0.0^98  0.02*19  0.0148  0.0134  -  0.65^9 oMh  -  -  0.0057  -n  X  —J.  Cu  -3 rt>  T J CO cr 3  CU  <  r+  —'•  »—i  O  •  3  o  - h CD rt>  CO  O CQ  <<  3  Pectoral rays  ~3  c r Oi o T3 — '  es  3 "  r  —i.  cn  •  O  < CU  CU  ro  T  T"  CD  Taholah  ro  o  •o  <o  no  Copalis  —  Conner Creek  —  /TN  ft <£>  co 3  — / T N  CT rt>  Oyhut rt>  n  c+  O "5  Humptulips  —  Wishkah  —  Wynoochee  —  Cu  -tt 3  -3  CU v< cn  3 "  SZ  CT cr  cn  (TJ  o  Satsop  •o 0  -51;. l a s t group was made up o f H u m p t u l i p s , Wishkah, Wynoochee, and Satsop f i s h w i t h a combined mean o f 20.52 r a y s .  P e l v i c f i n rays (Tables 19, 20; F i g u r e V I I I The ANOVA F - s t a t i s t i c c o u l d not be used due t o i t s l a c k o f agreement w i t h t h e Welch and Brown-Forsythe t e s t s .  A non-parametric K r u s k a l l -  W a l l i s t e s t showed no d i f f e r e n c e s i n mean c o u n t s . P e l v i c f i n r a y s were an almost i n v a r i a t e c h a r a c t e r , most f i s h having seven r a y s . v a r i a t i o n was f i n r a y l o s s r e s u l t i n g i n s i x r a y s .  The o n l y  L e s s e r f i n r a y counts  comprised a v e r y small f r a c t i o n , 6% o r l e s s , o f t h o s e c o l l e c t i o n s i n which v a r i a t i o n was found.  The Taholah sample was noteworthy s i n c e 3 out  o f t h e 19 f i s h (16%) had 6 p e l v i c f i n r a y s .  S c a l e rows b e f o r e t h e d o r s a l f i n o r i g i n  ( T a b l e s 2 1 , 22; F i g u r e IX)  With t h e e x c e p t i o n o f comparisons w i t h t h e Wishkah sample, a l l c o l l e c t i o n s were s t a t i s t i c a l l y equal and had a combined mean o f 21.38 s c a l e rows.  Wishkah specimens  averaged 23.82 s c a l e rows, s i g n i f i c a n t l y  d i f f e r e n t at t h e p = 0.001 l e v e l  from a l l  o t h e r samples.  On a v e r a g e ,  Wishkah f i s h had 2.02 s c a l e rows more than _N. hubbsi from o t h e r 1ocalities. Lateral  row s c a l e s (Tables 23, 24; F i g u r e X)  T a h o l a h , Oyhut, and Humptulips samples  had s t a t i s t i c a l l y equal  means, w i t h a combined average o f 50.80 s c a l e s .  The SNK i n d i c a t e d t h a t  s c a l e counts f o r t h e s e p o p u l a t i o n s averaged l e s s than t h e means f o r Wynoochee and Satsop c o l l e c t i o n s , t h e l a t t e r  statistically  i n d i s t i n g u i s h a b l e from each o t h e r w i t h a combined mean o f 52.21 s c a l e s . N o n - s i g n i f i c a n c e between Taholah and Satsop sample means may have been  Table 19. Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r t h e number o f p e l v f i n rays i n N. h u b b s i . Number o f p e l v i c f i n rays 95% c o n f i dence i nterval  6.  _7  _N  Logarithmic mean  Tahol ah  3  16  19  0.8345  0.0252  6.84  6.64 - 7.03  Copal i s  2  55  57  0.8428  0.0126  6.96  6.91 - 7.02  53  53  0.8451  0.0000  7.00  -  Location  Conner Creek  s.d.  Arithmetic mean  Oyhut  1  63  64  0.8441  0.0087  6.98  6.94 - 7.02  Humptuli ps  1  33  34  0.8431  0.0117  6.97  6.90 - 7.03  82  82  0.8451  0.0000  7.00  -  39  40  0.8434  0.0109  6.97  6.92 - 7.03  68  68  0.8451  0.0000  7.00  Wishkah Wynoochee Satsop  1  53  T a b l e 20. A n a l y s i s of v a r i a n c e and K r u s k a l l - W a l l i s t e s t s on t h e number of p e l v i c f i n rays i n _N. h u b b s i . The data f o r t h e s e t e s t s a r e p r e s e n t e d i n Table 19. The Levene as w e l l as t h e Brown-Forsythe and Welch t e s t s i n d i c a t e d t h a t t h e a n a l y s i s o f v a r i a n c e F - s t a t i s t i c s h o u l d not be used, so data was a n a l y z e d w i t h a non-parametric K r u s k a l l - W a l l i s t e s t . No d i f f e r e n c e s between p o p u l a t i o n s were i n d i c a t e d by t h e l a t t e r t e s t .  of  Source Variation  Sums o f Squares  df  Mean Squares  Total  0.0293  416  Groups  0.0021  7  0.0003  Error  0.0272  409  0.0001  F = 3.00 F 0.0005(1), 7,409 = 2.96; r e j e c t n u l l Levene's t e s t f o r equal v a r i a n c e s = 19.13; p < 0.001 One-way a n a l y s i s of v a r i a n c e , t e s t s t a t i s t i c s f o r w i t h i n - g r o u p v a r i a n c e s not assumed t o be e q u a l : Welch=0.52; p < 0.70 Brown-Forsythe=2.22 p < 0.12 Note - Only t h o s e groups w i t h non-zero v a r i a n c e were used i n t h e computations o f t h e Levene, Welsh and Brown-Forsythe T e s t s . K r u s k a l l - W a l l i s , H=8.58; X a c c e p t nul1  2  0.05(7)=14.067,  TI -i. tQ C -) rt)  (V X T3 — CU 3 cu 1  3 i—c  CD to CD << O 3 to c r -5 O Cu —'TJ  Pelvic rays  to z r O)  o  I  < cu  Taholah  -«J  CO  7  —  o 3  Copalis  c  Conner  Creek  —  Oyhut  —  Humptulips  —  e  m u  T3  <  2  Wishkah  0 o  to  Wynoochee c cr cr to  CO  (t>  cu 3  Satsop  —  —  0 c  T a b l e 21. Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r t h e number o f s c a l e rows b e f o r e t h e d o r s a l f i n o r i g i n i n N. hubbsi. 'R' i s t h e o r d e r i n g o f l o g a r i t h m i c means a c c o r d i n g t o i n c r e a s i n g magnitude and i d e n t i f i e s t h e p o p u l a t i o n s as used i n t h e Student-Newman-Keuls t e s t (R, T a b l e 22b).  Number o f s c a l e rows before t h e d o r s a l f i n o r i g i n  Location  18 19 20 21 22 23 24 25 26 27 4  2  2  4 25 14 14  4  1  6  1  6  Taholah Oyhut  1  5  Humptul i p s  Satsop  7 11  1  2 12 23 23 12  Wi shkah Wynoochee  4  5  1  6  6  1 12 19 19 12  1  4  4 11  8  3  3  1  7  Arithmetic mean  95% confidence interval  _N  Logarithmic mean  s.d.  19  1.3300  0.0222  21.42  20.85 - 21.91  3  64  1.3192  0.0264  20.89  20.54 - 21.17  1  34  1.3285  0.0288  21.35  20.82 - 21.80  2  82  1.3763  0.0263  23.82  23.47 - 24.10  6  40  1.3327  0.0329  21.57  21.00  - 22.04  4  68  1.3360  0.0273  21.72  21.34 - 22.01  5  R_  cn  56  T a b l e 22. S t a t i s t i c a l a n a l y s e s u s i n g l o g a r i t h m i c means, t e s t i n g f o r i n t e r - p o p u l a t i o n a l d i f f e r e n c e s i n t h e number o f s c a l e rows b e f o r e t h e d o r s a l f i n o r i g i n i n _N. hubbsi: (a) a n a l y s i s o f v a r i a n c e and (b) S t u d e n t Newman-Keuls (SNK) f o r p a i r w i s e comparisons. 'R' i n t h e SNK i d e n t i f i e s t h e p o p u l a t i o n means i n T a b l e 2 1 ; s i g n i f i c a n c e l e v e l s a r e denoted by s u p e r s c r i p t a s t e r i s k s ( 0.0001=***).  a.  Analysis of variance  f o r d a t a i n T a b l e 21  Source of V a r i a t i o n  Sums of Squares  Total  0.3768  Groups  0.1447  Error  0.2322  Mean Squares  df 306 5  0.0289  301  0.0008  F = 36.13 F 0.0005(1) 5,301 = 4.56, r e j e c t n u l l Levene's t e s t f o r equal v a r i a n c e s = 0.90; p < 0.50 One-way a n a l y s i s o f v a r i a n c e , t e s t s t a t i s t i c s f o r w i t h i n - g r o u p v a r i a n c e s not assumed t o be e q u a l : Welch = 39.88; p < 0.001 Brown-Forsythe = 36.28; p < 0.001 b.  Student -Newmani - Keuls t e s t f o r d a t a i n T a b l e 21 R  1  2  3  4  5  x  1.3192  1.3285  1.3300  1.3327  1.3360  N  19  64  34  82  40  R  X  N  1  1.3192  19  2  1.3285  64  0.0093  3  1.3300  34  0.0108  0.0015  4  1.3327  82  0.0135  0.0042  0.0027  5  1.3360  40  0.0168  0.0075  0.0060  0.0033  6  1.3763  68  0.65^1  0.8*75  0.5^3  0.8$3"6  -  F i g u r e IX. Geographic v a r i a t i o n d o r s a l f i n o r i g i n i n N. h u b b s i . symbols.  i n the number of s c a l e rows b e f o r e t h e See F i g . I I f o r an e x p l a n a t i o n of  Table 23. Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r t h e number o f l a t e r a l row s c a l e s i n NL hubbsi. 'R' i s t h e o r d e r i n g o f l o g a r i t h m i c means a c c o r d i n g t o i n c r e a s i n g magnitude and i d e n t i f i e s t h e p o p u l a t i o n s as used i n t h e Student-Newman-Keuls t e s t (R, Table 24b). Number o f l a t e r a l Location  46  Humptuli ps  48  2  Tahol ah Oyhut  47  1  1  4  1  2  49  50  51  52  53  54  row s c a l e s 55  56  57  7  2  3  2  2  12  11  17  4  6  2  2  7  8  1  2  4  2  2  8  9  12  20  15  Wi shkah  59  60  61  62  63  _N 19  1  7  58  3  64  1  34 3  8  4  1  82  Wynoochee  2  2  5  5  6  16  1  1  1  1  40  Satsop  1  4  5  17  11  10  10  4  3  3  68  Table 23.  Continued  Location  Logarithmic mean  s.d.  Arithmetic mean  Taholah  1.7055  0.0172  50.79  49.81  - 51.72  1  Oyhut  1.7065  0.0191  50.92  50.31  - 51.44  3  Humptuli ps  1.7057  0.0177  50.82  50.07 - 51.50  2  Wishkah  1.7485  0.0163  56.56  55.58 - 56.50  6  Wynoochee  1.7159  0.0161  52.02  51.39 - 52.60  4  Satsop  1.7189  0.0112  52.38  52.01  5  c o n f i dence i nterval  - 52.69  R_  cn vo  60  T a b l e 24. S t a t i s t i c a l a n a l y s e s u s i n g l o g a r i t h m i c means, t e s t i n g f o r i n t e r p o p u l a t i o n a l d i f f e r e n c e s i n t h e number o f l a t e r a l s c a l e s i n JN. hubbsi: (a) a n a l y s i s o f v a r i a n c e and (b) Student-Newman-Keuls (SNK) f o r p a i r w i s e comparisons. 'R' i n t h e SNK i d e n t i f i e s t h e p o p u l a t i o n means i n T a b l e 23; s i g n i f i c a n c e l e v e l s a r e denoted by s u p e r s c r i p t a s t e r i s k s (0.05=*; 0.01=**; 0.001=***).  a.  Analysis of variance  t a b l e f o r d a t a i n Table 23  Source of V a r i a t i o n  Sums o f Squares  df  Mean Squares  Total  0.1802  306  Groups  0.0893  5  0.0179  Error  0.0909  301  0.0003  F = 59.27 F 0.0005(1) 5,301 = 4.56; r e j e c t n u l l Levene's t e s t f o r equal v a r i a n c e s = 0.35; p > 0.85 One-way a n a l y s i s o f v a r i a n c e , t e s t s t a t i s t i c s f o r w i t h i n - g r o u p v a r i a n c e s not assumed t o be e q u a l : Welch = 60.27; p < 0.001 Brown-Forsythe = 58.61; p < 0.001 b.  Student-Newman-Keuls t e s t f o r data i n Table 23 R  R  X  1  X  1.7055  n  19  3  4  5  6  1.7065  1.7159  1.7189  1.7485  64  68  40  82  2 1.7057 34  n  -  1  1.7055  19  2  1.7057  34  0.0002  3  1.7065  64  0.0010  0.0008  4  1.7159  68  0.0104  0.0102  0.§094  5  1.7189  40  0.0t35  0.M32  0.0l2"4  6  1.7485  82  0.8130 oMh  -  oMh  0.0030  -  5  7  r  62  56  5 5  5 41 CO CO  o  CO  «  5 3  40  CD  68  +->  CO  0  52 19 64  51  34  4> 4>  50  F i g u r e X. Geographic v a r i a t i o n i n the number o f l a t e r a l hubbsi. See F i g . I I f o r an e x p l a n a t i o n o f symbols.  row  seal  62 due t o t h e small sample s i z e o f t h e former c o l l e c t i o n s i n c e d i f f e r e n c e s between Oyhut and Satsop as w e l l as Humptulips and Satsop were a r i t h m e t i c a l l y - l e s s but t e s t e d as s i g n i f i c a n t l y d i f f e r e n t . the  Means from  T a h o l a h , Oyhut, H u m p t u l i p s , Wynoochee and Wishkah samples were each  statistically  d i f f e r e n t from t h e Wishkah mean a t t h e p = 0.001  level.  The Wishkah mean was 5.28 and 3.87 s c a l e s g r e a t e r , r e s p e c t i v e l y , than t h e above combined means. S c a l e s above t h e l a t e r a l Statistically-equal  s c a l e row ( T a b l e s 25, 26; F i g u r e XI)  C o p a l i s and Conner Creek means were  a r i t h m e t i c a l l y g r e a t e r than o t h e r sample mans f o r c o l l e c t i o n s made west of t h e Wynoochee s i t e , a l t h o u g h t h i s d i f f e r e n c e was not s t a t i s t i c a l l y significant.  The T a h o l a h , Oyhut, Humptulips and Wishkah means were  statistically  equal.  and Satsop c o u n t s .  These f o u r , i n t u r n , averaged l e s s t h a n Wynochee The combined mean count f o r T a h o l a h , Oyhut,  Humptulips and Wishkah f i s h was 9.40 s c a l e s i n c o n t r a s t t o 9.81 s c a l e s for  Wynoochee and Satsop  fish.  S c a l e s below t h e l a t e r a l  s c a l e row ( T a b l e s 27, 28; F i g u r e X I I )  N e a r e s t - n e i g h b o r sample means were not s t a t i s t i c a l l y However, means i n c r e a s e d c l i n a l l y  different.  from Taholah and Oyhut, w i t h a combined  mean o f 9.25 s c a l e s , t o Wishkah, which averaged 9.71 s c a l e s . from Wynoochee f i s h were s t a t i s t i c a l l y  Mean counts  equal t o t h o s e from Wishkah_N.  h u b b s i ; however, means from Satsop f i s h were l e s s than t h o s e from  Wishkah  ones but e q u i v a l e n t t o t h o s e from Wynoochee specimens.  Lateral  bars i n males  ( T a b l e s 29, 30; F i g u r e X I I I , XIV)  The Wynoochee and Satsop sample means were s i g n i f i c a n t l y  different  Table 25. Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r t h e number o f s c a l e s above t h e l a t e r a l s c a l e row i n H. hubbsi. 'R' i s t h e o r d e r i n g o f l o g a r i t h m i c means a c c o r d i n g t o i n c r e a s i n g magnitude and i d e n t i f i e s t h e p o p u l a t i o n s as used i n t h e Student-Newman-Keuls t e s t (R, Table 26b). Number o f s c a l e s above l a t e r a l row  L o c a t i on  0.9686  0.0219  9.32  9.08 - 9.53  2  38  0.9795  0.0231  9.55  9.37 - 9.71  5  28  50  0.9798  0.0230  9.56  9.40 - 9.69  6  40  21  63  0.9678  0.0246  9.30  9.15 - 9.42  1  18  16  34  0.9757  0.0232  9.47  9.28 - 9.63  4  34  41  79  0.9754  0.0280  9.46  9.31 - 9.59  3  6  34  40  0.9931  0.0164  9.85  9.72 - 9.96  8  14  52  68  0.9898  0.0223  9.78  9.65 - 9.89  7  Tahol ah  13  6  19  Copali s  17  21  Conner Creek  22  Humptulips Wishkah  4  Wynoochee Satsop  1  R  s.d.  10  2  95% c o n f i dence i nterval  Arithmetic mean  9  Oyhut  Logarithmi c mean  11  1  N  cn  64  T a b l e 26. S t a t i s t i c a l a n a l y s e s u s i n g l o g a r i t h m i c means, t e s t i n g f o r i n t e r p o p u l a t i o n a l d i f f e r e n c e s i n t h e number o f s c a l e s above t h e l a t e r a l s c a l e row i n _N. h u b b s i ; (b) a n a l y s i s o f v a r i a n c e and (b) Student-Newman-Keuls (SNK) f o r p a i r w i s e comparisons. 'R' i n t h e SNK i d e n t i f i e s t h e p o p u l a t i o n means i n T a b l e 25; s i g n i f i c a n c e l e v e l s a r e denoted by s u p e r s c r i p t a s t e r i s k s ( 0.05=*; 0.01=**; 0.001=***). a.  Analysis  o f v a r i a n c e f o r data i n T a b l e 25 Source of V a r i a t i o n  Sums o f Squares  Mean Squares  df  Total  0.2221  390  Groups  0.0269  7  0.0038  Error  0.1952  383  0.0005  F = 7.60 F 0.0005(1) 7,383 = 3.84; r e j e c t n u l l Levene's t e s t f o r equal v a r i a n c e s = 9.56; p < 0.001 One-way a n a l y s i s o f v a r i a n c e , t e s t s t a t i s t i c s f o r w i t h i n - g r o u p v a r i a n c e s not assumed t o be equal Welch = 11.17; p < 0.001 Brown-Forsythe = 10.08; p < 0.001 b.  Student;-Newman-Keuls t e s t f o r R  1  2  X  0.9678  0.9686  n  63  19  data i n Tablei 25 4  5  6  7  8  0.9757  0.9795  0.9798  0.9898  0.9931  34  38  50  68  40  3 0.9754 79  n  R  X  1  0.9678  63  2  0.9686  19  0.0008  3  0.9754  79  0.0076  0.0068  4  0.9757  34  0.0079  0.0071  0.0003  5  0.9795  38  0.0117  0.0109  0.0041  0.0038  6  0.9798  50  0.0120  0.0112  0.0044  0.0041  0.0003  7  0.9898  68  oMn  0.8144  0.ol41  0.0103  0.0100  8  0.9931  40  0.6245  0.8177  0.0174  0.o!36  0.6133 0.0033  -  -  -  -  -  -  -  0)  o CO  o ©10  g  19  3B  0} CO CD _  - 2 O  to  CD o  ~ CO  40  50  34 63  68  79  L  U  I  JC CO  I co  a  o  O  I CO CO  CJ cu  c c  o  I Z3 SZ  O  I  co  a a  E D  I SZ  CO  JC co  I  CU  cu SZ  o o o  c >>  I  a  o co  CO  CO  o  Figure XI. Geographic v a r i a t i o n in the number of scales above the l a t e r a l scale row i n _N. hubbsi. F i g . II for an explanation of symbols.  See  Table 27. Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r t h e number o f s c a l e s below the l a t e r a l s c a l e row i n _ N . hubbsi. 'R' i s t h e o r d e r i n g o f l o g a r i t h m i c means a c c o r d i n g t o i n c r e a s i n g magnitude and i d e n t i f i e s t h e p o p u l a t i o n s as used i n t h e Student-Newman-Keuls t e s t (R, T a b l e 28b). Number o f s c a l e s below l a t e r a l row  19  0.9534  0.0285  9.00  7.83 - 10.31  1  2  65  0.9687  0.0270  9.32  8.21 - 10.53  3  10  2  27  0.9757  0.0318  9.48  8.13 - 10.99  4  33  37  8  79  0.9861  0.0308  9.71  8.41 - 11.15  6  22  17  1  40  0.9759  0.0251  9.48  8.42 - 10.63  5  48  19  68  0.9663  0.0220  9.26  8.36 - 10.23  2  10  Taholah  3  13  3  Oyhut  2  42  19  Humptulips  1  14  Wi shkah Wynoochee 1  R_  s.d.  9  Satsop  Arithmetic mean  Logarithmic mean  8  L o c a t i on  95% c o n f i dence i nterval  11  12  1  N  67  T a b l e 28. S t a t i s t i c a l a n a l y s e s u s i n g l o g a r i t h m i c means, t e s t i n g f o r i n t e r p o p u l a t i o n a l d i f f e r e n c e s i n t h e number o f s c a l e s below t h e l a t e r a l s c a l e row i n H. hubbsi: (a) a n a l y s i s o f v a r i a n c e and (b) Student-Newman K e u l s (SNK) f o r p a i r w i s e comparisons. 'R' i n t h e SNK i d e n t i f i e s t h e p o p u l a t i o n means i n Table 29; s i g n i f i c a n c e l e v e l s a r e denoted by s u p e r s c r i p t a s t e r i s k s (0.05=*; 0.01=**; 0.001=***).  a.  A n a l y s i s o f v a r i a n c e t a b l e f o r d a t a i n Table 27 Source of V a r i a t i o n  Sums o f Squares  df  Mean Squares  Total  0.2433  297  Groups  0.0256  5  0.0051  Error  0.2177  292  0.0007  F = 7.29 F 0.0005(1) 5,292 = 4.57; r e j e c t n u l l Levene's t e s t f o r equal v a r i a n c e = 5.62; p < 0.001 One-way a n a l y s i s o f v a r i a n c e , t e s t s t a t i s t i c s f o r w i t h i n group v a r i a n c e s not assumed t o be e q u a l : Welch = 6.91; p < 0.001 Brown-Forsythe = 6.75; p < 0.001 b.  Student-Newman-Keuls t e s t f o r d a t a i n Table 27 R  R  X  1  X  0.9534  n  19  2 0.9663  3  4  5  0.9687  0.9757  0.9759  65  27  40  68  n  -  1  0.9534  19  2  0.9663  68  0.0129  3  0.9687  65  0.0153  0.0024  4  0.9757  27  0.0223  0.0094  0.0070  5  0.9759  40  0.02*25  0.0096  0.0072  0.0002  6  0.9861  79  oMb  0.8198 0.6174  0.0104  -  0.0102  10  79  o 19  CO CO 9 Q)  40  65  68  e  I CO (0 _ O CD  (0  CO  I o CO  O  I  I  I  a  ca  CL)  a E  10  3  X  O  o o c ><  D. O in  ca CO  F i g u r e X I I . Geographic v a r i a t i o n i n t h e number of s c a l e s below t h e l a t e r a l See F i g . I I f o r an e x p l a n a t i o n of symbols.  s c a l e row i n _N- hubbsi  T a b l e 29. Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r t h e number o f l a t e r a l bars i n male H. h u b b s i . 'R' i s t h e o r d e r i n g o f l o g a r i t h m i c means a c c o r d i n g t o i n c r e a s i n g magnitude and i d e n t i f i e s t h e p o p u l a t i o n s as used i n t h e Student-Newman Keuls t e s t (R, Table 13b). Number o f l a t e r a l bars  L o c a t i on  9_ 10  Tahol ah  4  2  Copali s Oyhut  7  Humptuli ps Wishkah  2  11  12  3  3  4  8  4  2  1  11  13  10  7  5  1  3  5  5  11 .12  12  13  Wynoochee Satsop  1  2  14  15  16  17  18  19  Logarithmi c ' N mean  s .d.  Arithmetic mean  95% confidence interval  R_  12  1.0149  0.0520  10.41  9.59-11.17  1  19  1.0906  0.0384  12.37  11.81-12.85  3  1  54  1.0876  0.0553  12.33  11.82-12.66  2  1  1  16  1.1224  0.0394  13.31  12.64-13.90  5  17  2  2  1  59  1.1059  0.0508  12.84  12.37-13.16  4  1  1  5  14  1  22  1.2193  0.0211  16.59  16.21-16.92  7  13  10  7  8  2  44  1.1825  0.0420  15.29  14.78-15.68  6  1  70  Table 30. S t a t i s t i c a l a n a l y s e s u s i n g l o g a r i t h m i c means t e s t i n g f o r i n t e r p o p u l a t i o n a l d i f f e r e n c e s i n t h e number o f l a t e r a l bars i n male N. h u b b s i ; (a) a n a l y s i s o f v a r i a n c e and (b) Student-Newman-Keuls (SNK) f o r p a i r w i s e comparisons. 'R' i n t h e SNK i d e n t i f i e s t h e p o p u l a t i o n means i n Table 29; s i g n i f i c a n c e l e v e l s a r e denoted by s u p e r s c r i p t a s t e r i s k s ( 0.001=***). a.  A n a l y s i s o f v a r i a n c e f o r d a t a i n Table 29 Source of V a r i a t i o n  Sums o f Squares  Mean Squares  df  Total  1.0876  225  Groups  0.6676  6  0.1113  Error  0.4200  219  0.0019  F = 58.58 F 0.0005(1) 6,219 = 4.19; r e j e c t n u l l Levene's t e s t f o r equal v a r i a n c e s = 3.84; p < 0.003 One-way a n a l y s i s o f v a r i a n c e , t e s t s t a t i s t i c s f o r w i t h i n - g r o u p v a r i a n c e s not assumed t o be e q u a l : Welch = 52.82; p < 0.001 Brown-Forsythe = 42.00; p < 0.001 b.  Student -Newman -Keuls t e s t f o r data i n Table 29 R  R  X  3  2  1  4  5  6  X  1.0149  1.0876  1.0906  1.1059  1.1224  1.1825  N  12  54  19  59  16  44  N  -  1  1.0149  12  2  1.0876  54  0.0*27  3  1.0906  19  0.8857  4  1.1059  59  *** 0.0910  5  1.1224  16  o.Ws  6  1.1825  44  7  1.2193  22  o.fele  0.0030  -  0.0183  0.0153  oMfo  0.85*8 0.8319  0.0165  0.6*66  0.8*01  0.f»7  o.!!54  0.8*>*.9  0M%9  -  7/  17 r  16h 44  15h  14 h  16  CO k-  C3  JO  «k_  59 1  3  » -  19 54  CD  CO  0  12  12  11  ioH  9 »-  I  I  I  I  CO  .c  a o  CO  JO  o .c to  t-  a o O  O  3  3  1 0  1  5  ^  n  U  m  b  CO  o o o  co  E  "! "  CD  .c  Q.  X  "ZbbsT^SPP^^T'f" "' *  IN. nuDbsi.  .a 3  CO  CO  CO  c >>  5  6  r  °  f  l  a  t  See F i g . I I f o r an e x p l a n a t i o n o f symbols.  6  r  a  l  b  a  r  S  i  n  m  a  l  e  I 0  I  I  1  2  centimeters  F i g u r e XIV. Male N. hubbsi from f o u r d i f f e r e n t r e g i o n s : (a) t h e O u i n a u l t R. d r a i n a g e , (b) t h e Wishkah R. d r a i n a g e , ( c ) trie Satsop R. d r a i n a g e and (d) t h e Wynoochee R. d r a i n a g e . Note t h a t t h e specimens from west of t h e Montesano H i l l s ( a , b) have l e s s e r numbers of l a t e r a l bars than t h o s e f i s h c o l l e c t e d east of t h e Montesano H i l l s ( c , d ) . The Q u i n a u l t R. male was c a p t u r e d at a c l o s e d h a b i t a t w h i l e a l l o t h e r s were o b t a i n e d from open ones.  -3 5 0  73  from each o t h e r at t h e p = 0.001 and 15.29  bars, respectively.  l e v e l , w i t h average bar counts o f  T h i s was  c o n t r a s t e d by s i g n i f i c a n t l y  bar means from c o l l e c t i o n s made e l s e w h e r e .  differentiate. bars.  level.  statistically  The t h r e e c o l l e c t i o n s had a combined mean of  The Wishkah mean, 12.84  lower  The Taholah sample mean  t e s t e d as l e s s than a l l o t h e r mean bar counts at t h e p = 0.001 C o p a l i s , Oyhut and Humptulips means were not  16.59  12.51  b a r s , was s i g n i f i c a n t l y g r e a t e r than means  from C o p a l i s , Oyhut and Humptulips f i s h at t h e p = 0.001  level.  A west  t o e a s t i n c r e a s e i n bar numbers o c c u r r e d from c o a s t a l l o c a l i t i e s t o t h e Wishkah s i t e , t h i s having been 2.43  bars i n magnitude.  An  abrupt  i n c r e a s e i n mean counts was found between t h e Wishkah and Wynoochee s i t e s , almost 4 bars i n magnitude,  a l t h o u g h t h e two c o l l e c t i n g s i t e s  had  been i n c l o s e r p r o x i m i t y than were t h e o t h e r s , 16 km compared t o an average o f a p p r o x i m a t e l y 26  km.  Chromosome c h a r a c t e r i s t i c s No obvious d i f f e r e n c e s were observed i n comparisons  between males  and females or between p o p u l a t i o n s i n chromosome numbers o r morphology. A summary by l o c a t i o n of _N. hubbsi examined i n t h e chromosomal i s p r e s e n t e d i n Table 31.  A total  analysis  o f 84 males and 77 females were  examined f o r chromosome numbers but o n l y 32 males and 30 females were used i n s o r t i n g and q u a n t i f y i n g m i t o t i c s p r e a d s .  In t h e s e l a t t e r c a s e s ,  3 k a r y o t y p e s per i n d i v i d u a l were e v a l u a t e d , w i t h c a r e h a v i n g been t a k e n t o use e q u a l l y as w e l l as not t o o c o n t r a c t e d chromosomes. females were examined from a l l  Both males and  c o l l e c t i n g s i t e s , w i t h two e x c e p t i o n s .  Only males were c o l l e c t e d at Hanaford Creek and o n l y one female from t h e J u n c t i o n C i t y l o c a l i t y was  karyotyped.  74  T a b l e 31. The c a t e g o r i e s of N. hubbsi used i n t h e chromosome a n a l y s i s . "Number examined" r e f e r s t o tRbse f i s h from which k a r y o t y p e s were o b t a i n e d ; "number q u a n t i f i e d " r e f e r s t o t h a t subset o f t h e "number examined" f o r which 3 s o r t e d k a r y o t y p e s were measured.  Males Location Tahol ah Oyhut Humptuli ps Wi shkah Junction  City  Wynoochee Satsop Black  River  Hanaford Total  Creek  Number exami ned  Females Number quantified  Number exami ned  Numbe r quanti f i e d  5  3  5  3  21  12  20  8  6  2  5  2  20  4  17  6  -  -  1  1  8  3  5  3  20  4  20  5  2  2  4  2  2  2  -  -  84  32  77  30  75 The  frequency  d i s t r i b u t i o n of chromosome numbers i n g i l l  c e l l s f rom _N. hubbsi 32.  c o l l e c t e d at d i f f e r e n t s i t e s i s presented  The modal d i p l o i d count was  chromosomes were p r o b a b l y  48 chromosomes.  epithelial i n Table  Counts g r e a t e r than  48  t h e r e s u l t s of m i t o t i c n o n d i s j u n c t i o n , and  chromosome breakage or s e p a r a t i o n as w e l l as chromosomes o r i g i n a t i n g from o t h e r m i t o t i c c e l l s ; hypomodal counts were l i k e l y a consequence of chromosome l o s s d u r i n g sample p r e p a r a t i o n . C e n t r i c f u s i o n s and f i s s i o n s have been found r e s p o n s i b l e f o r an i n t e r t i s s u e chromosomal polymorphism i n the s a l m o n i d , (Ohno et a l . , 1965). somatic  S_. i r i d e u s Gibbons  Most s t u d i e s on f i s h chromosomes have i n v o l v e d o n l y  chromosome spreads.  The polymorphism i n ^.  irideus indicates  t h a t f u s i o n s and f i s s i o n s c o u l d be r e s p o n s i b l e f o r d i f f e r e n c e s i n chromosome number between somatic Since i n t r a i n d i v i d u a l  and gonadal c e l l s f o r o t h e r  polymorphisms would be d e t e c t a b l e as d i f f e r e n c e s i n  chromosome number, somatic  d i p l o i d counts s h o u l d be compared w i t h m e i o t i c  ones t o determine whether somatic of gonadal ones.  fishes.  chromosome spreads are r e p r e s e n t a t i v e  Metaphase I I chromosome s e t s from t e s t e s c o n s i s t e n t l y  y i e l d e d 24 chromosomes w h i l e metaphase I spreads had 48 ( F i g . 15). frequency  d i s t r i b u t i o n of m i t o t i c counts f o r c e l l s from f i n , g i l l ,  i n t e s t i n e and kidney are p r e s e n t e d number was  The  i n Table 33.  S i n c e t h e modal  48 chromosomes f o r a l l t i s s u e s and t h i s number was  diploid  t h e same as  observed f o r Metaphase I t e s t i c u l a r c e l l s as w e l l as two times t h a t seen in  metaphase I I c e l l s , _N. hubbsi  was  considered  i n v a r i a t e regarding  i n t r a i n d i v i d u a ! polymorphisms i n chromosome number. d i f f e r e n t t i s s u e sources but o n l y g i l l  c o u l d have been i n t e r c h a n g e d  e p i t h e l i u m was  M i t o t i c c e l l s from f o r my a n a l y s i s ,  used i n t h e remainder of t h i s a n a l y s i s .  A summary o f chromosome morphometries i s g i v e n i n Table 34.  A  76  Table 32. Frequency d i s t r i b u t i o n o f d i p l o i d chromosome numbers as o b t a i n e d from c e l l s i n d i f f e r e n t t i s s u e s o f _N. h u b b s i . The r e s u l t s o f f i n , i n t e s t i n e , and k i d n e y were combined from t h e a n a l y s i s o f 1 male and 1 female from each o f t h e f o l l o w i n g l o c a t i o n s : Oyhut, Wynoochee and S a t s o p . The r e s u l t s f o r g i l l i n c l u d e t h o s e t a b u l a t e d i n T a b l e 33.  Number o f chromosomes p e r m i t o t i c  spread  40  41  42  43  44  45  46  47  48  49  50  Fin  1  2  0  1  0  1  0  5  29  0  0  Gill  3  1  4  1  7  8  10  24  113  4  2  I n t e s t i ne  0  1  0  2  1  1  1  2  18  1  0  Kidney  0  1  0  2  3  1  3  3  28  0  1  Total  4  5  4  6  11  11  14  34  188  5  3  Tissue  ~7~J  F i g u r e XV. Metaphase chromosome spreads from N. h u b b s i : ( a ) second spermatogonia! d i v i s i o n showing 24 b i v a l e n t s , l b ) m i t o t i c complement from g i l l e p i t h e l i u m w i t h 48 chromosomes and ( c ) m i t o t i c complement from k i d n e y t i s s u e w i t h 48 chromosomes.  10/Lim  79  Table 33. Frequency d i s t r i b u t i o n o f chromosome counts from t h e g i l l e p i t h e l i u m o f _N. h u b b s i . Eleven spreads c o n t a i n i n g l e s s than 40 chromosomes a r e not p r e s e n t e d i n t h i s t a b l e . Note t h e n e g a t i v e skew o f t h e d i s t r i b u t i o n as w e l l as t h e 48 chromosome mode i n a l l c a s e s .  Location  Number of chromosomes p e r m i t o t i c  spread  40  41  42  43  44  45  46  47  48  49  50  51  Satsop  1  0  0  1  2  2  1  7  20  1  0  1  Wynoochee  0  0  1  0  0  2  3  5  18  0  1  0  Wi shkah  0  1  0  0  2  1  3  8  39  2  1  0  Oyhut  1  0  0  0  1  0  2  0  9  0  0  1  Taholah  0  0  1  0  1  1  0  3  12  0  0  0  Black River  0  0  0  0  1  2  1  1  15  1  0  0  All  1  0  2  1  7  8  10  24  113  4  2  2  sites  80  T a b l e 34. Morphometries o f t h e N. hubbsi chromosome complement based on 186 chromosome spreads o b t a i n e d Tro'm 32 males and 30 f e m a l e s .  Relative length Pair No.  (0/0 o f d i p l o i d s e t ) Mean  Arm r a t i o  S.D.  / s h o r t arm 1ong arm Mean  x  1 Q Q  \ S.D.  1  6.8  0.4  59.5  10.4  2  6.0  0.4  60.3  6.3  3  5.5  0.4  44.6  9.6  4  5.2  0.3  44.0  8.7  5  5.1  0.3  53.0  8.5  6  4.8  0.3  66.2  7.6  7  4.6  0.3  56.3  5.9  8  4.4  0.3  48.8  8.6  9  4.3  0.2  53.8  7.6  10  4.1  0.2  63.8  9.3  11  3.9  0.3  55.1  9.9  12  3.7  0.4  40.6  12.8  13  3.3  0.3  41.9  10.2  14  3.1  0.3  42.4  10.6  15  4.6  0.5  14.4  12.7  16  4.1  0.5  12.8  12.0  17  3.8  0.3  0  0  18  3.6  0.2  0  0  19  3.4  0.2  0  0  20  3.3  0.2  0  0  21  3.2  0.2  0  0  22  3.1  0.2  0  0  23  2.8  0.3  0  0  24  2.7  0.2  0  0  81  s o r t e d k a r y o t y p e i s p r e s e n t e d i n F i g u r e X V I , f o l l o w e d by a diagrammatic ideogram i n F i g u r e X V I I . chromosomal groups.  The d i p l o i d spread was s o r t a b l e i n t o t h r e e  Chromosomes i n which mean arm r a t i o s were submedian  o r s u b t e r m i n a l made up t h e f i r s t group, d e s i g n a t e d chromosome p a i r s 114.  The second one c o n s i s t e d o f t e r m i n a l  r e g i o n chromosomes, p a i r s 15  and 16, w h i l e t h e t h i r d c a t e g o r y c o n t a i n e d t e r m i n a l sensu s t r i c t o p a i r s 17-24.  ones,  W i t h i n each group t h e chromosomes were ranked by d e c r e a s i n g  mean r e l a t i v e l e n g t h s . Chromosomes 1 and 2 were u s u a l l y i d e n t i f i a b l e w i t h o u t s o r t i n g o r measuring  ( F i g s . XV and X V I I I ) .  Chromosomes 15 and/or 16 had v e r y s h o r t  l e s s e r arms a l t h o u g h t h e l a t t e r sometimes appeared absent i n m i t o t i c spreads from t h e same i n d i v i d u a l  ( F i g . X V I ) . When l e s s e r arms were  p r e s e n t , t h e s e chromosomes c o u l d a l s o be d i s c e r n e d w i t h o u t s o r t i n g o r measuring.  The r e m a i n i n g chromosomes r e q u i r e d some q u a n t i f i c a t i o n f o r  p r o p e r assignment.  Terminal sensu s t r i c t o chromosomes were r e a d i l y  i d e n t i f i e d as a group.  Short o r l e s s e r arms were never seen i n  chromosome p a i r s 17-24, even when chromosome spreads were very e l o n g a t e ( F i g . 19).  For t h e most p a r t , a s s i g n a t i o n o f chromosomes was not h a r d ,  s i n c e chromosome arms had f e a t u r e s , e u c h r o m a t i c and h e t e r o c h r o m a t i c r e g i o n s , t h a t helped t h e p a i r i n g and s o r t i n g p r o c e s s .  Measurements o f  chromosomes f u r t h e r a i d e d i d e n t i f i c a t i o n . The number o f chromosome arms i s r e f e r r e d t o as t h e fundamental number.  T h i s term o r i g i n a t e d w i t h t h e assumption t h a t biarmed  chromosomes a r o s e from t h e f u s i o n o f uniarmed ones (Matthey, 1953), but t h e r e a l i z a t i o n t h a t o t h e r c y t o g e n e t i c phenomena ( e . g . , i n v e r s i o n s , c e n t r i c t r a n s p o s i t i o n s ; see J a c k s o n , 1975) may g e n e r a t e biarmed chromosomes has rendered t h e 'fundamental  number' a d e s c r i p t i o n of  F i g u r e XVI. S o r t e d karyotypes o f N. h u b b s i : (a) from a male c o l l e c t e d a t Oyhut and (b) a female c a p t u r e d i n the Satsop R i v e r d r a i n a g e . Both m i t o t i c complements were o b t a i n e d from g i l l e p i t h e l i a l cells. Note t h a t chromosomes 15 and 16 do not have l e s s e r arms i n (a) but do have them i n ( b ) . The presence o r absence o f these l e s s e r arms does not r e p r e s e n t as i n t e r s e x u a l o f i n t e r p o p u l a t i o n a l polymorphism. R a t h e r , chromosome spreads from a s i n g l e i n d i v i d u a l can have e i t h e r s t a t e s .  c -o _i o  roO  4-  r —  cu  2  3  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  Chromosome Number  F i g u r e X V I I . Idiogram o f m i t o t i c metaphase chromosomes from N. h u b b s i , based on t h e measurements o f arm r a t i o and r e l a t i v e l e n g t h . T h i s f i g u r e was d e r i v e d from the q u a n t i f i c a t i o n o f 186 chromosome spreads from 32 males and 30 females.  OO  %5  F i g u r e X I I X . Metaphase chromosome spreads from the g i l l e p i t h e l i u m o f N. hubbsi c a p t u r e d a t v a r i o u s l o c a t i o n s : (a) Q u i n a u l t R i v e r male, (b) Oyhut f e m a l e , ( c ) Humptulips R i v e r male, (d) Wishkah R i v e r f e m a l e , (e) Hanaford Creek male and ( f ) Satsop R i v e r female (used t o c o n s t r u c t the k a r y o t y p e i n F i g . XVIb). Chromosomes 1 and 2 a r e r e l a t i v e l y easy to d i s c e r n i n u n s o r t e d chromosome spreads ( e . g . , t h o s e chromosomes denoted by arrows i n b, e and f ) . The chromosomes spreads i n c and d a r e e n l a r g e d more than the o t h e r ones. The spread o b t a i n e d from the Wishkah R i v e r female (d) i s more c o n t r a c t e d than i s the complement from the Humptulips R i v e r male ( c ) . E x c e s s i v e c o n t r a c t i o n r e s u l t s i n an accompanying l o s s o f chromosomal d e t a i l s , w i t h d i f f e r e n t chromosome p a i r s t e n d i n g to be s i m i l a r i n the more c o n t r a c t e d s p r e a d s . Such p r e p a r a t i o n s were not used f o r chromosomal a n a l y s i s i n t h i s s t u d y . •  85a  F i g u r e XIX. from a male out some o f chromosomes of levan et to p a i r s 17  A m i t o t i c spread comprised o f e l o n g a t e chromosomes, o b t a i n e d N. hubbsi c o l l e c t e d i n t h e Wishkah R. d r a i n a g e . Arrows p o i n t those chromosomes a p p e a r i n g d e v o i d o f l e s s e r arms. These a r e c o n s i d e r e d t e r m i n a l sensu s t r i c t o u s i n g t h e t e r m i n o l o g y a l . (1964). Terminal sensu s t r i c t o chromosomes a r e a s s i g n e d - 24 i n s o r t e d karyotypes ( F i g s . XVI and X V I I ) .  87 c e n t r o m e r i c p o s i t i o n (White, 1978; Swanson e t a l . , 1981).  For _N. h u b b s i ,  t h e fundamental number was a s c e r t a i n e d t o be 80 chromosomal arms p e r diploid  karyotype.  DNA q u a n t i f i c a t i o n Feulgen-DNA d e t e r m i n a t i o n s were done on e r y t h r o c y t e n u c l e i from f i v e male and f i v e female _N. hubbsi 35).  f o r each o f f o u r c o l l e c t i n g s i t e s  (Table  A t - t e s t i n d i c a t e d no i n t e r s e x u a l d i f f e r e n c e s i n DNA v a l u e s and an  ANOVA r e v e a l e d no i n t e r p o p u l a t i o n a l d i f f e r e n c e s ( T a b l e 3 6 ) .  Feulgen-DNA  d e t e r m i n a t i o n s were based on r a t i o s between t h e d e n s i t i e s o f f i s h cell  n u c l e i and t h o s e from c h i c k e n e r y t h r o c y t e s .  blood  R a t i o data u s u a l l y  f o l l o w s a b i n o m i a l d i s t r i b u t i o n and s h o u l d be t r a n s f o r m e d t o a r c s i n e s f o r a near-normal d i s t r i b u t i o n ( Z a r , 1974).  My data were c o n v e r t e d t o  a r c s i n e s i n o r d e r t o permit t h e above p a r a m e t r i c a n a l y s e s .  The grand  mean a b s o l u t e Feulgen-DNA e s t i m a t e f o r H. hubbsi was 1.68 pg/nucleus  ±  0.02 ( S . E . ) . F l u o r o m e t r i c assays r e s u l t e d i n a b s o l u t e DNA e s t i m a t e s o f 1.5 and 1.6 pg/nucleus  (Table 3 5 ) .  Feulgen-DNA e s t i m a t e .  These were 11 and 7% l e s s than t h e mean  P r e v i o u s i n v e s t i g a t o r s ( e . g . , M i k s c h e , 1971;  Mizuno and Macgregor, 1974) have r e p o r t e d s i m i l a r d i s c r e p a n c i e s when comparing b i o c h e m i c a l and m i c r o d e n s i t o m e t r i c DNA measurements. Feulgen-DNA d e t e r m i n a t i o n i s an assay on aldehydes  The  of deoxyribose  sugars  (Sharma and Sharma, 1972) w h i l e t h e f l u o r o m e t r i c one i s a q u a n t i f i c a t i o n of p u r i n e n u c l e o t i d e s ( F o s t e r and Gurney, 1976).  D i f f e r e n c e s between t h e  Feulgen and f l u o r o m e t r i c r e s u l t s c o u l d have been because each o f t h e two methods i n v o l v e d a d i f f e r e n t aspect o f t h e DNA m o l e c u l e . c o u l d have been miscounted  f o r t h e f l u o r o m e t r i c assay.  Erythrocytes Hinegardner  88  Table 35. D e t e r m i n a t i o n s o f n u c l e a r DNA c o n t e n t s i n t h e e r y t h r o c y t e s o f H. hubbsi: (a) F e u l g e n - d e n s i t o m e t r y and (b) f l u o r o m e t r i c d e t e r m i n a t i o n s f o r two females from t h e Satsop R. s i t e . A l t h o u g h o n l y two f i s h were p r o c e s s e d i n t h e f l u o r o m e t r i c a s s a y , t h e r e s u l t a n t DNA e s t i m a t e s were c o n s i d e r e d c l o s e enough t o t h e Feulgen ones t o a t t e s t t o t h e r e l i a b i l i t y of t h e l a t t e r .  a.  Feulgen-densitometry Rel a t i ve  T  Location  _N  DNA  s.d.  Oyhut males females combined Wishkah males females combined  5 5 10  0.665 0.672 0.668  0.070 0.057 0.064  5 5 10  0.668 0.674 0.671  0.058 0.066 0.062  Wynoochee males females combined  5 5 10  0.671 0.673 0.672  0.052 0.063 0.058  Satsop males females combined  5 5 10  0.673 0.670 0.672  0.042 0.051 0.047  *DNA c o n t e n t r e l a t i v e t o c h i c k e n e r y t h r o c y t e s , w h i c h have 2.50 pg/nucleus ± 0.04 ( S . E . ) . i. Grand mean f o r r e l a t i v e Feulgen-DNA = 0.671 + 0.017 (S.E.) ii. Mean a b s o l u t e Feulgen-DNA e s t i m a t e u s i n g t h e grand mean f o r r e l a t i v e Feulgen-DNA = 1.68 p g / d i p l o i d c e l l ± 0.02 ( S . E . ) . b.  Fluorometric determinations Specimen 1. 1150000 e r y t h r o c y t e s / m m b l o o d ; 7.36 yg DNA f o r 4866000 e r y t h r o c y t e s i n sample 3  Specimen 2. 985000 e r y t h r o c y t e s / m m b l o o d ; 7.01 yg DNA f o r 3815000 e r y t h r o c y t e s i n sample 3  A b s o l u t e DNA e s t i m a t e s = 1.5 and 1.6 p g / e r y t h r o c y t e  nucleus  89  Table 36. S t a t i s t i c a l a n a l y s e s u s i n g t h e a r c s i n square r o o t t r a n s f o r m of r e l a t i v e Feulgen-DNA v a l u e s f o r t h e n u c l e i o f N. hubbsi e r y t h r o c y t e s : (a) a n a l y s i s o f v a r i a n c e t e s t i n g f o r d i f f e r e n c e s between p o p u l a t i o n s and (b) t - t e s t comparing male and female n u c l e a r DNA c o n t e n t s . S i n c e no i n t e r s e x u a l o r i n t e r p o p u l a t i o n a l DNA d i f f e r e n c e s were found, a s i n g l e mean a b s o l u t e DNA v a l u e , 1.68 peg/nucleus + 0.02 (S.E.) as d e r i v e d i n T a b l e 35, was c o n s i d e r e d r e p r e s e n t a t i v e o f a l l _N. h u b b s i .  a.  A n a l y s i s o f v a r i a n c e f o r data i n Table 35a Mean Squares  Sums o f Squares  df  Total  180.23  39  Groups  4.58  3  1.53  Error  175.65  36  4.88  Source of V a r i a t i o n  F = 0.31 F 0.05(1) 3,36 = 2.86; do not r e j e c t n u l l B a r t l e t t ' s t e s t f o r equal v a r i a n c e = 2.66 0.05,3 = 7.82; do not r e j e c t n u l l 2  b.  t - t e s t f o r data i n Table 35a t = 0.28 t 0.05,38 = 2.02; do not r e j e c t n u l l F-max t e s t f o r equal v a r i a n c e = 1.17 F 0.025,19,19 = 2.53; do not r e j e c t n u l l  (1968) found t h a t c o u n t i n g e r r o r s w i t h a hemocytometer were g r e a t enough to s i g n i f i c a n t l y  a f f e c t DNA e s t i m a t e s .  S i n c e f l u o r o m e t r i c measurements  were not e x t r e m e l y d i f f e r e n t from t h e F e u l g e n - d e n s i t o m e t r i c ones, t h e former were c o n s i d e r e d t o be an i n d i c a t o r o f t h e a c c u r a c y o f t h e l a t t e r .  91  DISCUSSION The  Junction City c o l l e c t i n g The  River. nights  site  J u n c t i o n C i t y c o l l e c t i n g s i t e was Only two _N. hubbsi were c a p t u r e d  day  red spawning c o l o r s .  w i t h an a l k a l i n e pH and  The 2-4  a month f o r f i v e months.  k i l o m e t e r t o t h e west.  a physiological intolerance to i t .  considered  t o be marginal  connection Meldrim  aversion to dissolved s a l t , Small  numbers o f _N.  o c c u r at J u n c t i o n C i t y because of s a l i n e c o n d i t i o n s .  C i t y s i t e was  of the  collecting trips  ppt d i s s o l v e d s a l t s suggested a  (1968) showed t h a t _N. hubbsi has a b e h a v i o r a l  may  1979  Several  as  presence of red morphs as w e l l as water  w i t h e s t u a r i n e Grays Harbor, about one  suggesting  trap  = one t r a p n i g h t ) as w e l l  _G. a c u l e a t u s males caught d u r i n g t h e March - May had  Chehalis  at J u n c t i o n C i t y a f t e r 150  (one b a i t e d t r a p submerged o v e r n i g h t  s e i n i n g and d i p n e t t i n g one  near the mouth of t h e  The  h a b i t a t f o r _N. hubbsi  hubbsi  Junction and  p o s s i b l y an o u t e r l i m i t of d i s t r i b u t i o n . Conditions  s i m i l a r t o t h o s e at J u n c t i o n C i t y may  t h e absence of_N.  hubbsi e l s e w h e r e .  McPhail  be r e s p o n s i b l e f o r  (1967) and Meldrim (1968)  have suggested t h a t s u i t a b l e Novumbra h a b i t a t s o c c u r r e d  along  the  s o u t h e r n shore of Grays Harbor i m m e d i a t e l y southwest of the J u n c t i o n C i t y c o l l e c t i n g s i t e , but gave no reasons f o r t h e seeming absence of _N. there.  hubbsi  Caged i n d i v i d u a l s s u r v i v e d s e v e r a l months i n swamps or marshes  w i t h i n the region (Meldrim,  1968), i n d i c a t i n g t h a t c o n d i t i o n s c o u l d be  l e a s t b r i e f l y s u i t a b l e f o r JN.  hubbsi.  The topography of streams and e x c e s s i v e s a l i n i t i e s c o u l d  be  at  92  r e s p o n s i b l e f o r t h e absence of t h i s f i s h .  Streams t r a v e r s e s t e e p  g r a d i e n t s from t h e W i l l a p a H i l l s almost t o t h e shores of Grays Harbor S. G e o l o g i c a l Survey map:  -  Hoquiam (1962): 1:250000 s c a l e ) ,  (U.  restricting  l o t i c waters p r e f e r r e d by _N. hubbsi c l o s e t o t h e Grays Harbor e s t u a r y . S a l i n e i n t r u s i o n s i n t o t h e s e marshes o r swamps because o f t h e i r p r o x i m i t y t o a marine  h a b i t a t may  p r o h i b i t long-term establishment of_N. hubbsi.  The J u n c t i o n C i t y c o l l e c t i n g s i t e was  suggested t o c o n t a i n few H. hubbsi  f o r t h e same reason.  Meristic  variation  A b i o t i c developmental 1961;  f a c t o r s can cause m e r i s t i c v a r i a t i o n  L i n d s e y and H a r r i n g t o n , 1972).  (Barlow,  C o n s e q u e n t l y , whether o r not  c h a r a c t e r s are under s t r i c t g e n e t i c c o n t r o l cannot be determined controlled laboratory studies.  without  S i n c e _N. hubbsi i n h a b i t s a s p e c i f i c  h a b i t a t w i t h i n a r e s t r i c t e d g e o g r a p h i c a r e a having a s i m i l a r c l i m a t e t h r o u g h o u t , i n t e r p o p u l a t i o n a l m e r i s t i c v a r i a t i o n c o u l d have been m o s t l y genetically-based. C l i m a t e c o u l d have a f f e c t e d m e r i s t i c c h a r a c t e r f r e q u e n c i e s .  The  h a b i t a t s of _N. hubbsi are r e g u l a r l y s u b j e c t e d t o summer d e s s i c a t i o n ( R u f f n e r , 1980).  M o r t a l i t y i n _N. hubbsi was  noted a t Oyhut and Satsop i n  J u l y 1979, when h a b i t a t s i z e s had been reduced and water c h e m i s t r y and temperatures  p o s s i b l y most s t r e s s f u l t o f i s h .  S e v e r a l dead f i s h were  found i n d r i e d - u p p o o l s and t h e r e m a i n i n g bodies of water had t h e l o w e s t pH's  r e c o r d e d d u r i n g t h e s t u d y , as low as 4.5, w i t h no d e t e c t a b l e oxygen  i n some.  Higher summer water t e m p e r a t u r e s , e s p e c i a l l y a t t h e  c o l l e c t i n g s i t e , may  have been m e t a b o l i c a l l y s t r e s s f u l .  Satsop  Individuals with  c e r t a i n c h a r a c t e r s t a t e s c o u l d have d i e d d u r i n g such p e r i o d s o f  93  e n v i r o n m e n t a l s t r e s s , due t o e i t h e r p o o r e r f i t n e s s o r chance. The p a t t e r n s i n t h e g e o g r a p h i c d i s t r i b u t i o n o f mean counts f o r lateral  bars and anal f i n rays i n males as w e l l as s c a l e s above t h e  lateral  s c a l e row f o r males and females combined may be a t t r i b u t a b l e t o  h i s t o r i c a l events.  C h a r a c t e r means were d i s t r i b u t e d i n a manner  i n d i c a t i n g a b a r r i e r t o gene f l o w between t h e Wishkah and Wynoochee Rivers.  L a t e r a l bar means i n c r e a s e d c l i n a l l y from c o a s t a l t o i n l a n d  s i t e s w i t h t h e l a r g e s t d i f f e r e n c e i n mean counts between t h e Wishkah and Wynoochee c o l l e c t i n g s i t e .  The magnitude o f t h i s i n c r e a s e was  a p p r o x i m a t e l y two t i m e s g r e a t e r than any o t h e r n e a r e s t - n e i g h b o r difference.  Anal f i n r a y counts i n males had a d i s t r i b u t i o n i n which  Wynoochee and Satsop specimens western s i t e s . lateral  had l o w e r mean counts than f i s h from more  A d i f f e r e n t p a t t e r n was seen w i t h s c a l e s above t h e  s c a l e row.  Wishkah specimens  and t h o s e from more western  had l o w e r mean s c a l e counts than d i d Wynoochee and Satsop ones.  sites The  former s e r i e s of c o l l e c t i o n s d i d not d i f f e r among t h e m s e l v e s , but t h e i r mean counts were l e s s than t h e s t a t i s t i c a l l y - e q u a l  Wynoochee and Satsop  ones. The Montesano H i l l s  ( F i g . 1) c o n s i s t o f s e v e r a l  h i l l s , 200 - 400 m  i n h e i g h t , o r i g i n a t i n g i n t h e n o r t h as f o o t h i l l s o f t h e Olympic  Mountains  and making up t h e e a s t and west s l o p e s , r e s p e c t i v e l y , o f t h e Wishkah and Wynoochee d r a i n a g e s .  The Montesano H i l l s end a b r u p t l y i n t h e s o u t h as  b l u f f s , 30 - 40 m i n h e i g h t , a l o n g t h e f i n a l  k i l o m e t e r s of t h e C h e h a l i s  R i v e r b e f o r e t h e l a t t e r e n t e r s Grays Harbor  ( p e r s o n a l o b s e r v a t i o n ; U. S.  G e o l o g i c a l Survey map: Aberdeen (1962), 1:62500 s c a l e ) .  Along t h i s lower  s e c t i o n o f t h e r i v e r , t h e low r e l i e f t e r r a i n s u i t a b l e f o r Novumbra h a b i t a t s was s c a r c e due t o t h e b l u f f s and l i t t l e o r no s u i t a b l e  94 ' updrainage  h a b i t a t appeared p r e s e n t because o f t h e steep s l o p e o f t h e  Montesano H i l l s scale).  (U. S. G e o l o g i c a l Survey map: Aberdeen (1962); 1:62500  Such topography would i n h i b i t o r prevent d i s p e r s a l o f _N. hubbsi  through the region.  T h i s f i s h was not c a p t u r e d more than 5 km from t h e  mouths o f t h e Wishkah and Wynoochee R i v e r s , a l t h o u g h c o l l e c t i o n s were made f u r t h e r up t h e d r a i n a g e s . C o n d i t i o n s w i t h i n t h e C h e h a l i s R i v e r may a l s o r e s t r i c t movements o f _N. hubbsi between t h e Wishkah and Wynoochee d r a i n a g e s .  Meldrim  (1968)  has documented t h e a v e r s i o n by _N. hubbsi t o s a l i n e c o n d i t i o n s .  Saline  waters  from Grays Harbor extend u p r i v e r past t h e Montesano H i l l s ,  p o s s i b l y making t h e lower p a r t of t h e r i v e r i n t o l e r a b l e t o _N. h u b b s i . Beverage and Swecker (1969) r e p o r t e d 10 ppt d i s s o l v e d s a l t s i n C h e h a l i s R i v e r water samples from about 6 km west of Montesano, a t t h e s o u t h e a s t c o r n e r o f t h e Montesano H i l l s , as w e l l as 2 ppt s a l t f o r water samples a t Montesano i t s e l f .  Beverage and Swecker (1969) a l s o found t h a t water  f l o w s i n t h e C h e h a l i s R i v e r a n n u a l l y f l u c t u a t e d between 20 and 75 m / s e c , c o n s i d e r a b l y more than t h e n e g l i g i b l e water movement i n t y p i c a l h a b i t a t s ( M e l d r i m , 1968).  Novumbra  Large p i s c i v o r o u s f i s h e s o c c u r i n t h e C h e h a l i s  R i v e r (Wydoski and Whitney, 1979), t h e s e p o s s i b l y p r e y i n g upon _N. hubbsi i f and when i t e n t e r s t h e system. The s e p a r a t i o n o f t h e range o f JN. hubbsi i n t o two g e o g r a p h i c a l l y i s o l a t e d r e g i o n s ( i . e . , c o a s t a l and Grays Harbor d r a i n a g e s ; and t h e i n l a n d C h e h a l i s R i v e r subdrainage) least the Pleistocene.  may have been i n e x i s t e n c e s i n c e at  S u i t a b l e h a b i t a t f o r _N. hubbsi seems t o have been  l e s s l i k e l y w i t h i n t h e Wishkah and Wynoochee d r a i n a g e s , s i n c e a s o u t h e r n l o b e o f t h e Olympic G l a c i e r extended i n t o t h e n o r t h e r n Montesano H i l l s (Heusser, 1964).  I c e melt from t h i s g l a c i a l  lobe l i k e l y  resulted i n  95  g r e a t e r stream f l o w s and c o u l d be a reason f o r p r e s e n t  streams  s i t u a t e d i n deep, narrow r a v i n e s (U. S. G e o l o g i c a l Survey map: (1962); The  being Aberdeen  1:62500). Puget Lobe of the Vashon G l a c i e r extended s o u t h o f t h e Juan de  Fuca S t r a i t s and formed a p e r i g l a c i a l ( M c P h a i l , 1967;  McKee, 1972).  i n c r e a s e d by d r a i n a g e  The  l a k e d r a i n e d by the C h e h a l i s  r e s u l t a n t l a r g e r i v e r f l o w was  further  from i c e - b l o c k e d Cascade Mountain v a l l e y s , making  C h e h a l i s R i v e r d i s c h a r g e g r e a t e r than the present R i v e r (McKee, 1972).  f l o w i n t h e Columbia  I f i c e - c o v e r e d Montesano H i l l s b l o c k e d  d i s p e r s a l of _N. hubbsi o n l y have o c c u r r e d  River  the  d u r i n g t h e P l e i s t o c e n e , e a s t - w e s t movements c o u l d  by means o f t h e C h e h a l i s R i v e r .  were p r o b a b l y t o o f a s t f o r _N. hubbsi  However, f l o w r a t e s  t o use t h e r i v e r as a passageway.  As an a s i d e t o t h i s s t u d y , s t a r c h gel e l e c t r o p h o r e s i s was  done w i t h  muscle e x t r a c t s from t e n _N. h u b b s i , f i v e from the Wishkah c o l l e c t i n g and  f i v e from the Wynoochee one.  migrated  site  Three of t h e f o u r assayed enzymes  t h e same d i s t a n c e on t h e gel r e g a r d l e s s of the g e o g r a p h i c o r i g i n  o f t h e sample.  However, i s o c i t r a t e dehydrogenase was  Wishkah Novumbra were f i x e d f o r a gene product  found t o d i f f e r .  t h a t migrated  further  a n o d a l l y than d i d i s o c i t r a t e dehydrogenase o b t a i n e d from Wynoochee specimens.  Although  the sample s i z e was  i s o c i t r a t e dehydrogenase may  s m a l l , the f i x e d d i f f e r e n c e s i n  have been i n d i c a t i v e of t h e f a u n a l  barrier  i n d i c a t e d by the m e r i s t i c c h a r a c t e r a n a l y s i s . The  r e s u l t s of analyses  on the o t h e r m e r i s t i c c h a r a c t e r s d i d not  c o n t r a d i c t t h e Montesano H i l l s b e i n g a f a u n a l b a r r i e r .  P e c t o r a l f i n ray  counts i n c r e a s e d from western t o e a s t e r n s i t e s , p o s s i b l y because o f gene f l o w and t h e r e s t r i c t i o n of p r e d a t o r - f r e e h a b i t a t s t o c o a s t a l r e g i o n s . Reductions i n t h e number o f p e l v i c f i n rays were a l s o most common i n  96  closed habitats. escape from Closed  Lower f i n ray numbers c o u l d make a f i s h l e s s a b l e t o  predators. s i t e s were small  i n comparison t o t h e open ones, and  movements i n t o or from t h e former were r e s t r i c t e d . s i z e s i n c l o s e d h a b i t a t s may significant effect.  E f f e c t i v e population  be small enough f o r chance events t o have a  Reductions i n the numbers of p e l v i c and p e c t o r a l f i n  rays c o u l d have been t h e r e s u l t of g e n e t i c d r i f t as proposed f o r t h e Death V a l l e y c y p r i n o d o n t s Sexual s e l e c t i o n may  by M i l l e r  (1948).  be s t r o n g i n c l o s e d systems due  h a b i t a t s i z e s and t h e l a c k of p r e d a t o r s .  Male_N. hubbsi are  d u r i n g t h e b r e e d i n g season (Hagen et a l . , 1972). t h e small areas c o m p r i s i n g  t o the  territorial  Spawning t e r r i t o r i e s i n  c l o s e d s i t e s c o u l d be at a premium, so  f e a t u r e s enhancing t h e p r o b a b i l i t y of s u c c e s s f u l a c q u i s i t i o n and of good spawning s i t e s as w e l l as a t t r a c t i o n of mates might be favored  ( W i l l i a m s , 1975).  small  Even i f c e r t a i n sexual  f i s h more v i s i b l e t o p r e d a t o r s ,  character  any defense  strongly  s t a t e s made  s e l e c t i o n f o r t h e s e c h a r a c t e r s would  have t o be compromised w i t h a n t i - p r e d a t o r a d a p t a t i o n  because of  not  the  absence of p i s c i v o r e s . Both male c o u r t s h i p and male-male a g g r e s s i v e e x p a n s i o n and  interactions involve  f l u t t e r i n g of the d o r s a l f i n (Hagen et a l . , 1972).  The  h i g h e s t modes and means f o r the number of d o r s a l f i n rays i n males were found i n males from c l o s e d s i t e s .  Greater  numbers o f d o r s a l f i n rays  may  i n c r e a s e t h e expanse of the d o r s a l f i n making t h e l a t t e r more apparent t o conspecifics. The  l o w e s t counts i n l a t e r a l  closed s i t e s . competition  and  bars were found i n males caught at  A l e s s e r number of bars may female a t t r a c t i o n .  be f a v o r e d  Lesser l a t e r a l  f o r male-male  bar counts may  have  97been a s s o c i a t e d w i t h broader bar w i d t h but t h e l a t t e r was Lesser  not measured.  bar numbers, as w e l l as g r e a t e r numbers of d o r s a l f i n r a y s ,  make male _N. hubbsi more conspicuous t o p r e d a t o r s .  could  I f t h i s were t r u e ,  f i s h from open systems would be e x p e c t e d t o have g r e a t e r numbers of lateral  bars and l e s s e r numbers of d o r s a l f i n r a y s .  t h e number of l a t e r a l areas.  The  sexual  c h o i c e and  b a r s , i n c r e a s i n g from t h e P a c i f i c c o a s t t o i n l a n d  h i g h e r mean d o r s a l f i n ray counts were i n specimens from t h e  P a c i f i c coastal The  A cline existed for  region. s e l e c t i o n hypothesis  preference  may  be t e s t a b l e by means of mate  t e s t s (see M c P h a i l , 1969).  Females o r i g i n a t i n g  from anywhere w i t h i n t h e range would be expected t o p r e f e r e n t i a l l y o r i e n t a t e towards or mate w i t h _N. hubbsi males having l o w e r numbers o f lateral  bars and  h i g h e r d o r s a l f i n ray c o u n t s .  If t e r r i t o r i a l  defense  were a f f e c t e d by b a r r i n g or d o r s a l f i n ray s t a t e s , male-male e n c o u n t e r s d u r i n g the b r e e d i n g season c o u l d be examined. counts but  Males w i t h l o w e r  h i g h e r d o r s a l f i n ray counts would be e x p e c t e d t o be  bar better  a b l e t o h o l d onto a s i t e . No g e o g r a p h i c p a t t e r n s on anal  of v a r i a t i o n were i n f e r r e d from t h e  analysis  f i n ray counts i n f e m a l e s , a l t h o u g h Satsop specimens d i d have  more r a y s , on a v e r a g e , than d i d Humptulips ones.  In m a l e s , anal f i n ray  counts d i d not have t h e same g e o g r a p h i c p a t t e r n as counts on d o r s a l f i n rays.  better associated  with  a g e o l o g i c a l b a r r i e r r a t h e r than open o r c l o s e d h a b i t a t s s u g g e s t i n g  that  anal  The  d i s t r i b u t i o n of anal  f i n rays may  dorsal f i n rays.  f i n ray means was  not be under t h e same ( s e x u a l ) s e l e c t i v e regime as No s t a t i s t i c a l  d i f f e r e n c e s were d i s c e r n e d  f o r the  g e o g r a p h i c d i s t r i b u t i o n d o r s a l f i n ray means i n f e m a l e s ; however, t h e d i s t r i b u t i o n of mean counts n u m e r i c a l l y  approximated t h e  pattern  9 8 -  s t a t i s t i c a l l y - a s c e r t a i n e d f o r males. males and females shared  This r e s u l t c o u l d be e x p e c t e d i f  some o f t h e genes f o r d e t e r m i n i n g  this  character  s t a t e , w i t h s e x - l i m i t e d a l l e l e s r e s p o n s i b l e f o r t h e dimorphism.  Although  b o t h sexes might have had a s i m i l a r p a t t e r n o f v a r i a t i o n , males would have e x p r e s s e d The  i t more s t r o n g l y .  o n l y s i g n i f i c a n t d i f f e r e n c e s found i n t h e a n a l y s i s on caudal f i n  ray means were C o p a l i s and Conner Creek ones t e s t i n g as g r e a t e r than t h o s e from Wynoochee o r Satsop. Satsop and C o p a l i s samples.  Anal  f i n r a y counts d i f f e r e d between t h e  The l a t e r a l  bar and p e c t o r a l f i n ray means  f o r C o p a l i s f i s h were t h e same as t h o s e o b t a i n e d f o r r e c e n t from o t h e r c o a s t a l d r a i n a g e s .  No s t a t i s t i c a l  collections  d i f f e r e n c e s i n t h e number  of anal f i n rays i n males were found i n comparisons between T a h o l a h , C o p a l i s , Conner Creek, and Oyhut. Whether o r not c h a r a c t e r s t a t e s i n C o p a l i s R i v e r and Conner Creek specimens were r e p r e s e n t a t i v e o f c u r r e n t p o p u l a t i o n s r e q u i r e s updated collections.  C o p a l i s _N. hubbsi  ones i n 1968. residential  had been caught i n 1947, Conner Creek  The Conner Creek swamp has s i n c e been d r a i n e d f o r  development (Whitney and Wydowski, 1979; personal  o b s e r v a t i o n ) and t h e C o p a l i s l o c a t i o n was not l i s t e d i n t h e U n i v e r s i t y o f Washington r e c o r d s .  Temporal changes i n m e r i s t i c s t a t e s a r e documented  f o r o t h e r f i s h e s ( e . g . , L i n d s e y , 1953; C h e r n o f f ,  1982), so c h a r a c t e r s i n  C o p a l i s and Conner Creek f i s h may have changed from t h e past t o t h e present. The  p a t t e r n o f g e o g r a p h i c v a r i a t i o n i n t h e number o f l a t e r a l  row  s c a l e s suggested by data i n Meldrim (1968) was not found i n my a n a l y s i s . P o s s i b l e reasons f o r t h e h i g h means f o r s c a l e rows b e f o r e t h e d o r s a l f i n o r i g i n and l a t e r a l  row s c a l e s a t t h e Wishkah c o l l e c t i n g s i t e a r e not  99 •' '  obvious.  Perhaps t h e r e are f a c t o r s t o which d e v e l o p i n g eggs o f _N. hubbsi  are h y p e r s e n s i t i v e .  Mean counts f o r both c h a r a c t e r s were about  h i g h e r than averages from e l s e w h e r e . demonstrated  10%  L i n d s e y and H a r r i n g t o n (1972) have  s i m i l a r v e r t e b r a l and, t o a l e s s e r e x t e n t , p e l v i c f i n ray  v a r i a t i o n produced  by d i f f e r e n t developmental  temperatures.  My  physical  and chemical d e t e r m i n a t i o n s of t h e water at Wishkah were not d e s i g n e d t o d e t a i l t h e a q u a t i c h a b i t a t d u r i n g egg development.  At t h e l e v e l  r e s o l u t i o n i n t h i s s t u d y , c o n d i t i o n s at t h e Wishkah s i t e d i d not d i f f e r e n t from t h o s e i n waters e l s e w h e r e . c o l l e c t i o n , t h e r e was dorsal f i n o r i g i n .  of appear  E x c e p t i n g t h e Wishkah  no g e o g r a p h i c v a r i a t i o n i n s c a l e rows b e f o r e t h e  I f t h e mean l a t e r a l  row s c a l e count f o r Wishkah f i s h  i s removed from t h e a n a l y s i s , r e m a i n i n g samples have t h e i r means d i s t r i b u t e d i n a manner parsimonious w i t h t h e Montesano H i l l s b e i n g a b a r r i e r t o gene f l o w .  Wynoochee and Satsop f i s h had e q u i v a l e n t means.  Counts were s t a t i s t i c a l l y - g r e a t e r than t h e r e s u l t s o b t a i n e d from c o l l e c t i o n s made west of t h e Montesano H i l l s . had s t a t i s t i c a l l y - e q u a l  Chromosomal  These l a t t e r  collections  means.  state  No g e o g r a p h i c v a r i a t i o n s i n chromosome number o r morphology were found.  My work agreed w i t h t h a t by Beamish e t a l . (1971) r e g a r d i n g t h e  d i p l o i d number, 48 chromosomes i n both s t u d i e s .  My fundamental  number  f o r _N. hubbsi d i f f e r e d from t h e one by Gold e t a l . (1980), 74 arms i n t h e i r r e p o r t c o n t r a s t i n g w i t h 80 i n mine. C o l c h i c i n e - i n d u c e d d i f f e r e n c e s i n chromosomal c o n d e n s a t i o n may been r e s p o n s i b l e f o r t h e d i s c r e p a n c y i n fundamental  number.  have  Thompson  (1979) c o n s i d e r e d t h i s t o be t h e case f o r arm number d i f f e r e n c e s between  ,  100  h i s k a r y o l o g i e s o f c i c h l i d f i s h e s and t h o s e produced by Ohno and A t k i n (1966).  E x c e s s i v e exposure t o c o l c h i c i n e can cause t h e s m a l l e r  and/or  more c o n t r a c t i l e l e s s e r arms t o become t o o small f o r r e s o l u t i o n w i t h a l i g h t m i c r o s c o p e ( S m i t h , 1965).  E l o n g a t e chromosome spreads would,  t h e r e f o r e , be e s s e n t i a l components o f any c y t o g e n e t i c s t u d y , s i n c e t h e s e would be t h e m i t o t i c or m e i o t i c s e t s l e a s t a f f e c t e d by a n t i m i t o t i c s . Gold e t a l . (1980) counted chromosome arms i n t h e s i n g l e c o n t r a c t e d m i t o t i c spread f o r _ N . hubbsi i n Beamish e t a l . (1971) t o o b t a i n t h e fundamental number.  My d e t e r m i n a t i o n was d e r i v e d from t h e enumeration of  186 e l o n g a t e chromosome s e t s .  The newer fundamental arm number does not  r e s o l v e q u e s t i o n s on umbrid o r e s o c o i d i n t e r r e l a t i o n s h i p s , t h e s e f i s h e s r e m a i n i n g a c o n f u s i n g c h r o m o s o m a l l y - d i v e r s e group as d i s c u s s e d by  Beamish  et a l . (1971).  Heteromorphy  of chromosomes 15 and  16  Chromosomes 15 and 16 were anomalous, a p p e a r i n g uniarmed i n some m i t o t i c spreads but biarmed i n o t h e r s .  The uniarmed and biarmed  chromosomal s t a t e s were found i n d i f f e r e n t chromosome spreads o b t a i n e d from t h e same f i s h .  I f l e s s e r arms were h i g h l y c o n t r a c t e d then t h e y  not have been r e s o l v a b l e under t h e l i g h t m i c r o s c o p e . biarmed chromosomes 15 and 16 f e l l  may  The l o n g arms of  w i t h i n s i z e ranges e q u i v a l e n t t o t h e  r e l a t i v e l e n g t h s of t e r m i n a l chromosomes 15 and 16, s u g g e s t i n g t h a t i n v e r s i o n o r c e n t r i c t r a n s p o s i t i o n were not r e s p o n s i b l e f o r t h e polymorphism. The polymorphism c o u l d have been caused by o r i e n t a t i o n s o f t h e s e l e s s e r arms.  White  (1973) has argued t h a t t r u e t e r m i n a l chromosomes do  not e x i s t s i n c e a l l e x p e r i m e n t a l l y - i n d u c e d ones are u n s t a b l e .  There  c o u l d be fundamental d i f f e r e n c e s between n a t u r a l and i n d u c e d  101 terminal  chromosomes, but White (1973) s t a t e d t h a t l e s s e r h e t e r o c h r o m a t i c arms were always p r e s e n t but not n e c e s s a r i l y r e s o l v a b l e w i t h a l i g h t microscope.  S i n c e small  l e s s e r arms c o n s i s t c h i e f l y o f p a r a c e n t r i c  h e t e r o c h r o m a t i n , h i g h l y r e p e t i t i v e DNA sequences s i m i l a r l y r e p e a t e d on t h e proximal  r e g i o n o f t h e major arm (Swanson e t a l . , 1981), t h e seeming  absence o f l e s s e r arms i n chromosomes 15 and 16 c o u l d r e s u l t o f DNA-DNA h y b r i d i z a t i o n .  have been t h e  John and Freeman (1975) have observed  t h a t l e s s e r arms on t e r m i n a l - a p p e a r i n g  chromosomes were sometimes f o l d e d  back onto t h e major arm d u r i n g m e i o s i s and m i t o s i s .  I f DNA sequences on  t h e l e s s e r arm were a l i g n i n g themselves w i t h s i m i l a r ones on t h e major arm,  t h i s would be v i s u a l l y - o b s e r v a b l e as b a c k f o l d i n g .  N u c l e a r DNA Previous q u a n t i f i c a t i o n of nuclear  DNA c o n t e n t s f o r _N. hubbsi as  w e l l as t h e o t h e r e s o c o i d s (Beamish e t a l . , (1971) may be t o o h i g h . Beamish et a l . (1971) used human buccal erythrocytes  from _N. h u b b s i .  epithelium  as t h e r e f e r e n c e t o  Buccal e p i t h l i u m c o n s i s t s m o s t l y o f  k e r a t i n i z e d dead c e l l s  (Bloom and F a w c e t t , 1968) not n e c e s s a r i l y having  t h e same DNA c o n t e n t .  The n u c l e i o f buccal  epithelial  c e l l s are  k a r y o r r h e c t i c o r p y c n o t i c , both s t a t e s i n v o l v i n g changes i n and l o s s e s o f DNA ( P h a r r , 1976; Frankel  e t a l . , 1970).  N u c l e i c a c i d c o n t e n t s would not  n e c e s s a r i l y be t h e same between c e l l s as w e l l as l e s s than i n l i v i n g cells.  A l t h o u g h Beamish e t a l . (1971) p u r p o r t e d l y  f o l l o w e d t h e methods  o f Ohno and A t k i n (1966) f o r DNA q u a n t i f i c a t i o n , t h e c o n t r o l f o r t h e s e l a t t e r workers was t h y r o i d e p i t h e l i u m , w h i c h c o n s i s t s o f l i v i n g (Bloom and F a w c e t t , 1968) f o r which DNA d e t e r m i n a t i o n s would be  cells  102 r e p r e s e n t a t i v e o f t h e d i p l o i d genome s i z e . Beamish et a l . (1971) c a l c u l a t e d a b s o l u t e amounts o f DNA assigning to t h e i r epithelial  cell  averaged from an u n s p e c i f i e d l i s t understanding  standard a n u c l e i c a c i d of p l a c e n t a l mammals.  t h a t such l i s t s a r e u s u a l l y i n c o m p l e t e  by  content  I t i s my  compilations  i n c l u d i n g v a l u e s w i t h o u t r e g a r d t o t e c h n i q u e or a c c u r a c y  (e.g., Shapiro,  1976); such compendia are meant t o be b i b l i o g r a p h i e s not t r u s t w o r t h y d a t a sets. was  The a b s o l u t e DNA  v a l u e a s s i g n e d t o human buccal e p i t h e l i a l  about 14% more than t h e average per c e l l  humans (Swanson et a l . , 1981).  content e s t a b l i s h e d f o r  Using t h i s v a l u e would have made t h e  e s t i m a t e s i n Beamish e t a l . (1971) t o o h i g h .  Nucleated  e r y t h r o c y t e s were chosen as r e f e r e n c e c e l l s i n my q u a n t i f i c a t i o n o f DNA  p g / c e l l +_ 0.04  M i r s k y and R i s , 1951; al.,  study.  v a r i a t i o n i n the  ( S . E . ) , n = 7:  L e s l i e , 1955;  DNA  chicken The  i n c h i c k e n e r y t h r o c y t e s has been repeated  several investigators with l i t t l e ( a v e r a g i n g 2.50  cells  by  determinations  Davidson e t a l . ,  1950;  Vendrely and V e n d r e l y , 1956;  1971), so t h e y were a w e l l - s u i t e d s t a n d a r d f o r DNA  Rasch et  comparisons i n _ N .  hubbsi. U s i n g a f l u o r o m e t r i c t e c h n i q u e , V e n d r e l y and Vendrely Knobloch e t a l . (1958) e s t a b l i s h e d 1.7 e s o c o i d , _E. l u c i u s .  The DNA  pg D N A / d i p l o i d nucleus f o r an  v a l u e s from my  study were i n c l o s e agreement  w i t h t h i s d e t e r m i n a t i o n p o s s i b l y i n d i c a t i n g t h e DNA hubbsi  (1953) and  e s t i m a t e f o r H.  as having been a c c u r a t e .  The  n u c l e i c a c i d q u a n t i f i c a t i o n s of Beamish et a l . (1971) were  o f f e r e d e a r l i e r as s u p p o r t i n g e v i d e n c e f o r p o l y p i o i d a l salmonid  genome.  r e l a t i v e DNA  R e v i s i o n s i n a b s o l u t e DNA  o r i g i n of the  v a l u e s i f one  accepts  amounts from Beamish e t a l . (1971) obscure t h i s n o t i o n .  103 Using my a b s o l u t e Feulgen-DNA e s t i m a t e f o r _N. h u b b s i , t h e D a l l i a Novumbra-Esox group would have 26-37% o f t h e n u c l e a r DNA found i n t h e c e l l s o f salmonids  r a t h e r than a p p r o x i m a t e l y  F l u c t u a t i n g environmental DNA c o n t e n t s  50%.  regimes may s e l e c t f o r d i f f e r e n t  heritable  ( D u r r a n t , 1962), w i t h more extreme o r v a r i a b l e s i t u a t i o n s  a s s o c i a t e d w i t h h i g h e r n u c l e i c a c i d amounts.  F o r example, a c l i n e f o r  n u c l e a r DNA has been r e p o r t e d i n t h e gymnosperm, P i c e a s i t c h e n s i s , i n c r e a s i n g from n o r t h e r n C a l i f o r n i a t o t h e Kenai ( M i k s c h e , 1971).  Perhaps no i n t e r p o p u l a t i o n a l DNA d i f f e r e n c e s were  i n _ N . hubbsi s i n c e t h i s f i s h does not encounter environmental  Peninsula of Alaska found  a s i g n i f i c a n t amount o f  h e t e r o g e n e i t y ; i t o c c u r s i n a very p a r t i c u l a r h a b i t a t  w i t h i n a r e g i o n having a s i m i l a r c l i m a t e  throughout.  General d i s c u s s i o n A l t h o u g h v a r i a t i o n i n some e x t e r n a l m e r i s t i c c h a r a c t e r s was c o n s i d e r a b l e between p o p u l a t i o n s o f _N. h u b b s i , obvious d i f f e r e n c e s were not found i n chromosome morphology and number as w e l l as n u c l e a r content.  Congruence i n t h e p a t t e r n s o f m o r p h o l o g i c a l  DNA  and chromosome  v a r i a b i l i t y w i t h i n a s p e c i e s has been documented f o r i n s e c t s ( A t c h l e y , 1981).  Among f i s h e s , a l l o p a t r i c morphotypes o f t h e p o e c i l i i d ,  a f f i n i s , have been found w i t h d i f f e r e n t chromosome morphologies and H o w e l l , 1979), y e t _N. hubbsi  Gambusia (Black  from e i t h e r s i d e o f t h e Montesano H i l l s  appeared a l i k e r e g a r d i n g n u c l e a r parameters. c y p r i n o d o n t i d s o f t h e genus Cyprinodon  S i m i l a r t o _N. h u b b s i ,  have been c h a r a c t e r i z e d by a l a c k  of obvious chromosomal d i f f e r e n c e s but c o n s i d e r a b l e m o r p h o l o g i c a l differentiation N. hubbsi  (Turner and L i u , 1977; Stevenson,  1981).  i s found w i t h i n a temperate r a i n f o r e s t , and t h e general  104 r e g i o n i n which t h i s animal o c c u r s has l i k e l y c o n t a i n e d r a i n f o r e s t s i n c e t h e e a r l y Eocene ( W o l f e , 1981). which f o s s i l (Cavender,  The John Day d e p o s i t i n Oregon from  Novumbra have been r e c o v e r e d had a r a i n f o r e s t  origin  1969), s u g g e s t i n g t h a t t h i s f i s h has e x i s t e d w i t h i n t h e same  environment  s i n c e a t l e a s t t h e Oligocene epoch.  Conservatism i n  chromosome morphology and DNA c o n t e n t may be a consequence o f Novumbra having p e r s i s t e d i n t h e same environment  f o r several m i l l i o n years.  One  outcome o f t h i s may have been t h e e v o l u t i o n o f an optimum k a r y o t y p e and DNA c o n t e n t . With f i t n e s s r e g a r d i n g chromosomal s t a t e and DNA c o n t e n t  maximized,  f u r t h e r e v o l u t i o n a r y changes would o c c u r p r e d o m i n a t e l y t h r o u g h means o t h e r than a l t e r a t i o n s i n genome s i z e and r e l a t i v e gene p o s i t i o n and Baker, 1979).  (Bickham  The p r i m a r y amino a c i d sequence o f a p r o t e i n i s  determined by a s i n g l e gene and some d i f f e r e n c e s i n t h i s sequence may be d e t e c t e d by e l e c t r o p h o r e s i s .  Genie, o r e l e c t r o p h o r e t i c , c h a r a c t e r s i n _ N .  hubbsi were o n l y examined as an a s i d e .  The p i l o t e l e c t r o p h o r e t i c study  suggested g e o g r a p h i c v a r i a t i o n i n i s o c i t r a t e dehydrogenase, but f u r t h e r comments on g e n i e c h a r a c t e r s s h o u l d not be made u n t i l t h e c o m p l e t i o n o f substantial electrophoretic analyses.  M o r p h o l o g i c a l c h a r a c t e r s may v a r y  due t o d i f f e r e n c e s i n t h e i r r e g u l a t o r y c o n t r o l 1979).  (Bickham and Baker,  E x t e r n a l m o r p h o l o g i c a l d i f f e r e n c e s can be e x t e n s i v e between  p o p u l a t i o n s w i t h s i m i l a r k a r y o t y p e s (Swanson e t a l . , 1981) and would be an e x p e c t e d outcome o f g e o g r a p h i c i s o l a t i o n and s e l e c t i o n d i f f e r e n t morphological states i n d i f f e r e n t places.  favoring  External  m o r p h o l o g i c a l d i f f e r e n t i a t i o n i n _N. hubbsi o c c u r r e d as i n f e r r e d from my d a t a , i n response t o t h e presence o r absence o f p r e d a t o r s as w e l l as a l l o p a t r y due t o t h e Montesano H i l l s .  105  Chromosomal d i f f e r e n c e s w i t h i n _N. hubbsi may  e x i s t but t h e i r  d e t e c t i o n c o u l d be beyond t h e r e s o l u t i o n o f t h e G i e m s a - s t a i n i n g technique.  For example, p a r a c e n t r i c i n v e r s i o n s would have been  u n d e t e c t a b l e no m a t t e r how l a r g e t h e y were.  Inadequate  methodology,  t h e r e f o r e , may  have been p a r t l y r e s p o n s i b l e f o r t h e p e r c e i v e d chromosome  conservatism.  Intrachromosomal  s t a i n i n g t e c h n i q u e s may  s u i t a b l e f o r a n a l y z i n g chromosomal v a r i a t i o n . have had a slow development intrachromosomal  have been more  Such t e c h n i q u e s f o r f i s h e s  ( B l a x h a l l , 1975), u n l i k e t h e advances i n  r e s o l u t i o n a c h i e v e d w i t h chromosome spreads from h i g h e r  v e r t e b r a t e s (Hsu, 1973).  G-banding would have been most a p p r o p r i a t e  s i n c e almost every chromosome c o u l d be unambiguously  identified  chromosomal rearrangements t r a c e d (Bickham and Baker, 1976).  and  Attempts t o  G-band f i s h chromosomes have been u n s u c c e s s f u l so f a r (Denton, 1973;  Abe  and Muramoto, 1974; K l i g e r m a n and Bloom, 1976; Thorgaard, 1978; H a f e z , 1979; Gregory e t a l . , 1980). d i f f e r e n c e s i n _N. hubbsi may  Another way t o uncover chromosomal be t o examine m e i o t i c b i v a l e n t s i n h y b r i d s  between f i s h from d i f f e r e n t p o p u l a t i o n s ; t h e o n l y m e i o t i c spreads examined  i n my study were from w i l d - c a u g h t males f o r t h e purpose of  e s t a b l i s h i n g h a p l o i d chromosome numbers.  106  SUMMARY AND CONCLUSIONS 1.  N^. hubbsi has c o n s i d e r a b l e geographic v a r i a t i o n i n e x t e r n a l  m e r i s t i c c h a r a c t e r s caused by a l l o p a t r y , d i f f e r e n t i a l and  pressure,  sexual s e l e c t i o n . 2.  anal  predation  The geographic d i s t r i b u t i o n o f mean counts f o r l a t e r a l  f i n rays i n males as w e l l as s c a l e s above t h e l a t e r a l  bars and  s c a l e row f o r  males and females combined i n d i c a t e t h a t t h e Montesano H i l l s ,  those  f o o t h i l l s o f t h e Olympic Mountains s e p a r a t i n g t h e Wishkah R i v e r and Wynoochee R i v e r d r a i n a g e s , a r e a b a r r i e r t o gene f l o w .  The Montesano  H i l l s may have been such a b a r r i e r s i n c e a t l e a s t t h e P l e i s t o c e n e , s i n c e a l o b e o f t h e Olympic G l a c i e r extended i n t o t h i s this  region  during  g e o l o g i c a l epoch. 3.  The r e s u l t s r e g a r d i n g l a t e r a l  be a consequence o f sexual numbers o f l a t e r a l  s e l e c t i o n i n t h e absence o f p r e d a t o r s .  predator-free  habitats.  4.  These c h a r a c t e r  f o r mate a t t r a c t i o n and male-male a g g r e s s i v e  a c t i o n s , s i n c e d o r s a l f i n expansion and l a t e r a l behavioral  Lower  bars and h i g h e r d o r s a l f i n r a y counts i n males were  a s s o c i a t e d with small may be i m p o r t a n t  bar and d o r s a l f i n r a y numbers may  r e p e r t o i r e during breeding  The l a c k o f p r e d a t o r s  inter-  d i s p l a y s are part o f the  and t e r r r i t o r i a l  and small  trends  defense.  h a b i t a t s i z e may e x p l a i n t h e  tendency f o r p e c t o r a l and p e l v i c f i n r a y numbers t o be reduced i n small closed s i t e s .  Genetic  d r i f t c o u l d be i n c r e a s i n g t h e f r e q u e n c y o f f i s h  w i t h l e s s e r numbers o f p a i r e d f i n r a y s .  A l s o , t h e absence o f p r e d a t o r s  may r e l a x s e l e c t i o n f o r g r e a t e r numbers o f f i n rays i f t h e l a t t e r were i m p o r t a n t  f o r swimming speed and, hence, escape.  107 4.  The h i g h counts on l a t e r a l  row s c a l e s and s c a l e rows b e f o r e  the d o r s a l f i n o r i g i n i n f i s h from t h e Wishkah R i v e r d r a i n a g e may be due, i n p a r t , t o a b i o t i c developmental  factors.  S i n c e mean counts  a r e about 10% g r e a t e r than those from e l s e w h e r e , f u r t h e r  investigations  a r e n e c e s s a r y i n o r d e r t o p r o v i s i o n a l l y e x p l a i n t h e r e s u l t s i n a more s a t i f a c t o r y manner. 5.  N_. hubbsi appears t o be i n v a r i a t e i n t e r s e x u a l l y and geogra-  p h i c a l l y w i t h r e s p e c t t o chromosome number and gross morphology. the r e s u l t s o b t a i n e d by Beamish e t a l . ( 1 9 7 1 ) , 48 chromosomes the d i p l o i d number.  The fundamental  number determined  Like  comprise  i n t h i s study i s  80 chromosome arms, c o n t r a s t i n g w i t h 74 arms r e p o r t e d i n Gold e t a l . (1980).  The p r e s e n t s t u d y e n t a i l e d measurements on 186 e l o n g a t e c h r o -  mosome s e t s i n s t e a d o f t h e e v a l u a t i o n o f fewer c o n t r a c t e d ones. 6.  No d i f f e r e n c e s appear p r e s e n t i n t e r s e x u a l l y o r g e o g r a p h i c a l l y  r e g a r d i n g DNA c o n t e n t .  The Feulgen-DNA e s t i m a t e f o r e r y t h r o c y t e n u c l e i  i s about 1.68 p g / n u c l e u s , about 80% o f a p r e v i o u s l y - p u b l i s h e d v a l u e . T h i s d i s c r e p a n c y may be a t t r i b u t e d t o improper c o n t r o l c e l l s and a b s o l u t e DNA r e f e r e n c e v a l u e s b e i n g used i n t h e p r e v i o u s work.  S i n c e t h e Feulgen  d e t e r m i n a t i o n i s not v e r y d i f f e r e n t from my f l u o r o m e t r i c ones as w e l l as c l o s e t o v a l u e s a s c e r t a i n e d f o r a n o t h e r e s o c o i d , t h e DNA e s t i m a t e f o r H. hubbsi i s c o n s i d e r e d a c c u r a t e . 7.  The observed  c o n s e r v a t i s m i n chromosomal s t a t e and DNA c o n t e n t  c o u l d be a consequence o f N. hubbsi h a v i n g p e r s i s t e d i n a r a i n f o r e s t environment f o r s e v e r a l m i l l i o n y e a r s , perhaps s i n c e a t l e a s t t h e Oligocene.  An optimum karyotype and DNA c o n t e n t may have e v o l v e d .  108  LITERATURE CITED Abe, S., and J . Muramoto. 1974. D i f f e r e n t i a l s t a i n i n g o f chromosomes o f two s a l m o n i d s p e c i e s , S a l v e l i n u s leucomaenis ( P a l l a s ) and S a l v e l i n u s malma (Walbaum). P r o c . Jpn. Acad. 50:507-511. A t c h l e y , W. R. 1981. Chromosomal e v o l u t i o n and morphometric v a r i a b i l i t y i n t h e t h e l y t o k o u s i n s e c t Warramaba v i r g a (Key). Pages 371-397 U}_ W. R. A t c h l e y and D. S. Woodruff, eds. E v o l u t i o n and S p e c i a t i o n . Essays i n honor o f M. J . D. White. Cambridge U n i v e r s i t y P r e s s , Cambridge. B a r l o w , G. W. 1961. Causes and s i g n i f i c a n c e o f m o r p h o l o g i c a l i n f i s h e s . S y s t . Z o o l . 10:105-117.  variation  Beamish, R. J . , M. J . M e r r i l e e s , and E. J . Crossman. 1971. Karyotypes and DNA v a l u e s f o r members o f t h e s u b o r d e r E s o c o i d e i ( O s t e i c h t h y e s : S a l m o n i f o r m e s ) . Chromosoma ( B e r l . ) 34:436-447. B e r t o l l o , L. A. C , C. S. T a k a h a s h i , and 0. M. Fi 1 ho. 1979. K a r y o t y p i c s t u d i e s o f two a l l o p a t r i c p o p u l a t i o n s o f t h e genus H o p l i a s ( P i s c e s , E r y t h r i n i d a e ) . Rev. B r a s . Genet. I I 1:17-37. Beverage, J . P., and M. N. Swecker. 1969. E s t u a r i n e s t u d i e s i n upper Grays Harbor, Washington. U. S. G e o l o g i c a l Survey Water-Supply Paper 1873-B, 40 p. Bickham, J . W., and R. J . Baker. 1976. Chromosome homology and e v o l u t i o n o f emydid t u r t l e s . Chromosoma ( B e r l . ) 54:201-219. Bickham, J . W., and R. J . Baker. 1979. C a n a l i z a t i o n model o f chromosomal e v o l u t i o n . B u l l . C a r n e g i e Mus. Nat. H i s t . No. 13:70-84. B l a c k , A. and W. M. H o w e l l . 1978. A d i s t i n c t i v e chromosomal race o f t h e c y p r i n o d o n t i d f i s h , Fundulus n o t a t u s , from t h e Upper Tombigbee R i v e r system of Alabama and M i s s i s s i p p i . Copeia 1978:280-288. B l a c k , D. A. and W. M. H o w e l l . 1979. The North American m o s q u i t o f i s h , Gambusia a f f i n i s : a unique case i n sex chromosome e v o l u t i o n . Copeia 1979:509-513. B l a x h a l l , P. C. 1975. F i s h chromosome t e c h n i q u e s - a r e v i e w o f s e l e c t e d literature. J . F i s h B i o l . 7:315-320. Brown, M. B. and A. B. F o r s y t h e . 1974. Robust t e s t s f o r t h e e q u a l i t y o f v a r i a n c e s . J . Amer. S t a t i s t . Assoc. 69:364-367. Bush, G. L. 1975. 6:339-364.  Modes o f animal s p e c i a t i o n .  Bloom, W. and D. W. F a w c e t t . 1968. Saunders, P h i l a d e l p h i a ( P a . ) .  Annu. Rev. E c o l .  A Textbook of H i s t o l o g y .  Syst.  9 t h ed.  109  Bush, G. L., S. M. Case, A. C. W i l s o n and J . L. P a t t o n . 1977. Rapid s p e c i a t i o n and chromosomal e v o l u t i o n i n mammals. P r o c . Nat. Acad. S c i . USA 74:3942-3946. Cavender, T. 1969. An O l i g o c e n e mudminnow ( f a m i l y Umbridae) from Oregon w i t h remarks on r e l a t i o n s h i p s w i t h i n t h e E s o c o i d e i . Occ. Pap. Mus. Z o o l . Univ. M i c h . , No. 660:1-33. C h e r n o f f , B. 1982. C h a r a c t e r v a r i a t i o n among p o p u l a t i o n s and a n a l y s i s of biogeography. Am. Z o o l . 22:425-439. D a r l i n g t o n , P. J . , J r . 1957. d i s t r i b u t i o n of a n i m a l s .  the  Zoogeography. The g e o g r a p h i c a l W i l e y , New York (N.Y.).  D a v i d s o n , J . N., I . L e s l i e , R. M. S. S m e l l i e , and R. Y. Thomson. 1950. Chemical changes i n t h e d e v e l o p i n g c h i c k embryo r e l a t e d t o t h e d e o x y r i b o n u c l e i c a c i d c o n t e n t o f t h e n u c l e u s . Biochem. J . 46, Proc. XI. D e i t c h , A. D., D. Wagner, and R. M. R i c h a r t . 1968. Conditions i n f l u e n c i n g t h e i n t e n s i t y o f t h e Feulgen r e a c t i o n . J . Histochem. Cytochem. 16:371-379. Denton, T. E. 1973. F i s h Chromosome Methodology. S p r i n g f i e l d (111.).  C h a r l e s C. Thomas,  D i x o n , W. J . , M. B. Brown, L. Engelman, J . W. F r a n e , M. A. H i l l , R. I . J e n n r i c h , and J . D. Toporek, eds. 1981. BMDP S t a t i s t i c a l Software 1981. U n i v e r s i t y of C a l i f o r n i a P r e s s , B e r k e l e y ( C a l i f . ) D u r r a n t , A. Li num.  1962. The e n v i r o n m e n t a l H e r e d i t y 17:27-61.  i n d u c t i o n o f h e r i t a b l e changes i n  E n d l e r , J . A. 1977. Geographic V a r i a t i o n , S p e c i a t i o n and Princeton University Press, Princeton (N.J.).  Clines.  F i t z g e r a l d , J . W. 1957. Range e x t e n s i o n f o r t h e western mudminnow, Novumbra S c h u l t z . Copeia 1957:248. F o s t e r , D. N. and T. Gurney, J r . 1976. N u c l e a r l o c a t i o n of mammalian DNA polymerase a c t i v i t i e s . J . B i o l . Chem. 251:7893-7898. F r a n z , R. and D. S. Lee. 1976. A r e l i c t p o p u l a t i o n of t h e m o t t l e d s c u l p i n , Cottus b a i r d i , from t h e Maryland c o a s t a l p l a i n . Chesapeake S c i . 17:301-302. G o l d , J . R., W. J . K a r e l , and M. R. S t r a n d . 1980. Chromosome formulae of North American f i s h e s . P r o g r e s s i v e F i s h - c u l t u r i s t 42:10-23. Gregory, P. A., P. N. Howard-Peebles, R. D. E l l e n d e r and B. J . M a r t i n . 1980. A n a l y s i s o f a marine f i s h c e l l l i n e from a male sheepshead. J . Hered. 71:209-211.  110 H a f e z , R. 1979. A n a l y s e du c a r y o t y p e de l a tanche (Tinea t i n e a L.) par 1 ' o b t e n t i o n des bandes C e t G. Cymbium 3e s e r i e 7:15-26. Hagen, D. W., and G. E. E. Moodie. 1979. Polymorphism f o r b r e e d i n g c o l o r s i n G a s t e r o s t e u s a c u l e a t u s . I . T h e i r g e n e t i c s and geographic distribution. E v o l u t i o n 33:641-648. Hagen, D. W., G. E. E. Moodie and P. F. Moodie. 1972. T e r r i t o r i a l i t y and c o u r t s h i p i n the Olympic mudminnow (Novumbra h u b b s i ) . Can. J . Z o o l . 50:1111-1115. Hagen, D. W., G. E. E. Moodie and P. F. Moodie. 1980. Polymorphism f o r b r e e d i n g c o l o r s i n G a s t e r o s t e u s a c u l e a t u s . I I . R e p r o d u c t i v e success as a r e s u l t o f convergence f o r t h r e a t d i s p l a y . E v o l u t i o n 34:10501059. Heusser, C. J . 1964. P a l y n o l o g y o f f o u r bog s e c t i o n s from t h e western Olympic P e n i n s u l a , Washington. Ecology 45:23-40. H i n e g a r d n e r , R. T. 1968. E v o l u t i o n o f c e l l u l a r DNA c o n t e n t i n t e l e o s t f i s h e s . Am. Nat. 102:517-523. H i n e g a r d n e r , R. T. 1971. An improved f l u o r o m e t r i c assay f o r DNA. A n a l . Biochem. 39:197-201. Hoese, H. D. 1963. S a l t t o l e r a n c e o f the e a s t e r n mudminnow, Umbra pygmaea. Copeia 1963:165-166. Hsu, T. C. 1973. L o n g i t u d i n a l Rev. Genet. 7:153-176.  d i f f e r e n t i a t i o n o f chromosomes.  Annu.  Hubbs, C. L., and K. F. L a g l e r . 1964. F i s h e s o f t h e Great Lakes Region. U n i v e r s i t y o f . M i c h i g a n P r e s s , Ann A r b o r ( M i . ) . H u t c h i n s o n , G. E. 1957. New York (N. Y . ) .  A T r e a t i s e on Limnology.  Vol. I.  Wiley,  John, B., and M. Freeman. 1975. Causes and consequences o f R o b e r t s o n i a n exchange. Chromosoma ( B e r l . ) 52:123-130. Johnson, M. S., and M. F. M i c k e v i e h . 1977. V a r i a b i l i t y and e v o l u t i o n a r y r a t e s o f c h a r a c t e r s . E v o l u t i o n 31:642-648. K l i g e r m a n , A. D., and S. E. Bloom. 1976. D i s t r i b u t i o n o f F - b o d i e s , h e t e r o c h r o m a t i n , and n u c l e o l a r o r g a n i z e r s i n the genome o f the c e n t r a l mudminnow, Umbra 1 i m i . Cytogenet. C e l l Genet. 18:182-196. K l i g e r m a n , A. D., and S. E. Bloom. from s o l i d t i s s u e s o f f i s h e s . 2169.  1977. Rapid chromosome p r e p a r a t i o n s J . F i s h . Res. Board. Can. 34:2166-  Knobloch, A., C. V e n d r e l y , and R. V e n d r e l y . 1957. The amount o f d e s o x y r i b o n u c l e i c a c i d i n a s i n g l e t r o u t sperm. B i o c h i m . B i o p h y s . A c t a 24:201-202.  ' 111 LeGrande, W. H., and T. M. Cavender. 1980. The chromosome complement o f the s t o n e c a t madtom, Noturus f l a v u s ( S i l u r i formes: I c t a l u r i d a e ) , w i t h e v i d e n c e f o r the e x i s t e n c e o f a p o s s i b l e chromosomal r a c e . Copeia 1980:341-344. ?  L e l e k , A. 1980. Threatened f r e s h w a t e r f i s h e s o f Europe. C o u n c i l o f Europe ( S t r a s b o u r g ) , Nature and Environment S e r i e s , No. 18:269p. L e s l i e , I . 1955. The n u c l e i c a c i d c o n t e n t o f t i s s u e s and c e l l s . Pages 1-50 ijn E. C h a r g a f f and J . N. D a v i d s o n , eds. The N u c l e i c A c i d s . V o l . 2. Academic P r e s s , New York (N.Y.). L i n d s e y , C. C. 1953. V a r i a t i o n i n anal ray count o f the r e d s i d e s h i n e r Richardsonius balteatus (Richardson). Can. J . Z o o l . 31:211-225. L i n d s e y , C. C , and R. W. H a r r i n g t o n , J r . 1972. Extreme v e r t e b r a l v a r i a t i o n induced by temperature i n a homozygous c l o n e o f the s e l f - f e r t i l i z i n g c y p r i n o d o n t i d f i s h R i v u l u s marmoratus. Can. J . Zool . 50:733-744. Matthey, R. 1953. Les chromosomes des Muridae. R e v i s i o n c r i t i q u e e t m a t e r i a u x nouveaux pour s e r v i r a l ' h i s t o i r e de l ' e v o l u t i o n chromosomique chez ces Rongeurs. Rev. S u i s s e Z o o l . 50:225-283. M c L e i s h , J . , and N. S u n d e r l a n d . 1961. Measurement o f d e o x y r i b o s e n u c l e i c a c i d (DNA) i n h i g h e r p l a n t s by Feulgen photometry and chemical methods. Exp. C e l l Res. 24:527-540. McKee, B. 1972. Northwest.  Cascadia. The g e o l o g i c e v o l u t i o n o f the M c G r a w - H i l l , New York (N.Y.).  M c P h a i l , J . D. 1967. D i s t r i b u t i o n of freshwater Washington. Northwest S c i . 41:1-11. M c P h a i l , J . D. 1969. (Gasterosteus).  Pacific  f i s h e s i n western  P r e d a t i o n and the e v o l u t i o n o f a s t i c k l e b a c k J . F i s h . Res. Board Can. 26:3183-3208  M e l d r i m , J . W'. 1968. The e c o l o g i c a l zoogeography o f the Olympic mudminnow (Novumbra h u b b s i , S c h u l t z 1929). Ph.D. D i s s e r t a t i o n , U n i v e r s i t y o f Washington, S e a t t l e . M i k s c h e , J . P. 1971. I n t r a s p e c i f i c v a r i a t i o n o f DNA per c e l l between P i c e a s i t c h e n s i s (Bong.) C a r r . Provenances. Chromosoma ( B e r l . ) 32:343-352. M i l l e r , R. R. 1948. The c y p r i n o d o n t f i s h e s o f the Death V a l l e y system o f e a s t e r n C a l i f o r n i a and southwestern Nevada. M i s c . P u b l . Mus. Z o o l . Univ. Mich. 68:1-155. M i r s k y , A. E., and H. R i s . 1949. V a r i a b l e and c o n s t a n t components o f chromosomes. Nature (London) 163:666-667.  112  M i r s k y , A. E. and H. R i s . 1951. The d e s o x y r i b o n u c l e i c a c i d c o n t e n t of a n i m a l s and i t s e v o l u t i o n a r y s i g n i f i c a n c e . J . Gen. P h y s i o l . 34:451462. Mizuno, S. and H. C. Macgregor. 1974. Chromosomes, DNA sequences, and e v o l u t i o n i n salamanders of t h e genus P l e t h o d o n . Chromosoma ( B e r l . ) 48:239-296. Moodie, G. E. E. and T. E. Reimchen. 1976. P h e n e t i c v a r i a t i o n and h a b i t a t d i f f e r e n c e s i n G a s t e r o s t e u s p o p u l a t i o n s o f t h e Queen C h a r l o t t e i s l a n d s . S y s t . Z o o l . 25:49-61. M u r g a t r o y d , L. B. 1968. A q u a n t i t a t i v e i n v e s t i g a t i o n i n t o t h e e f f e c t o f f i x a t i o n t e m p e r a t u r e and a c i d s t r e n g t h upon t h e Feulgen r e a c t i o n . J . Roy. M i c r o s c o p . Soc. 88:133-144. Myers, G. S. 1951. F r e s h - w a t e r f i s h e s and East I n d i a n S t a n f o r d I c h t h y o l . B u l l . 4:11-21. Naiman, R. J . and D. L. S o l t z , eds. 1981. D e s e r t s . W i l e y , New York (N.Y.).  zoogeography.  F i s h e s i n North American  N e l s o n , G. J . and N. P l a t n i c k . 1981. S y s t e m a t i c s and Biogeography: c l a d i s t i e s and v i c a r i a n c e . Columbia U n i v e r s i t y P r e s s , New York (N.Y.). N i k o l s k y , G. 1976. The i n t e r r e l a t i o n between v a r i a b i l i t y o f c h a r a c t e r s , e f f e c t i v e n e s s of energy u t i l i z a t i o n , and k a r y o t y p e s t r u c t u r e i n f i s h e s . E v o l u t i o n 30:180-185. N i k o l s k y , G., and V. P. V a s i l y e v . 1973. On some r e g u l a r i t i e s i n t h e d i s t r i b u t i o n o f chromosome numbers i n f i s h . J . I c h t y o l . (Eng. T r a n s l . Vopr. I k h t i o l . ) 13:1-19. Ohno, S. and N. B A t k i n . 1966. Comparative DNA v a l u e s and chromosome complements o f e i g h t s p e c i e s of f i s h e s . Chromosoma ( B e r l . ) 18:456466. Ohno, S., C. S t e n i u s , E. F a i s s t , and M. T. Zenzes. 1965. Post-zygotic chromosomal rearrangements i n rainbow t r o u t (Salmo i r i d e u s , G i b b o n s ) . C y t o g e n e t i c s 4:117-129. O s t d i e k , J . L. and R. M. Nardone. 1959. S t u d i e s on t h e A l a s k a n b l a c k f i s h P a l l i a p e c t o r a l i s I . H a b i t a t , s i z e and stomach a n a l y s e s . Am. M i d i . Nat. 61:218-229. Peckham, R. S. and C. F. Dineen. 1957. Ecology o f t h e c e n t r a l mudminnow Umbia 1 imi ( K i r t l a n d ) . Am. M i d i . Nat. 58:222-231. P h a r r , S. L. 1970. I d e n t i f i c a t i o n o f benign and m a l i g n a n t c e l l s . Pages 996-1003 i n S. F r a n k e l , S. Reitman and A. C. S o n n e r w i r t h , eds. Gradwohl's C l i n i c a l L a b o r a t o r y Methods and D i a g n o s i s . 7 t h Ed. V o l . I. C. V. Mosby, S a i n t L o u i s (Mo.).  113  P i s t e r , E. P. 1981. The c o n s e r v a t i o n of d e s e r t f i s h e s . Pages 411-446 i n R. J . Naiman and D. L. S o l t z , eds. F i s h e s i n North American D e s e r t s . W i l e y , New York (N.Y.). Rasch, E. M., H. J . B a r r and R. W. Rasch. 1971. The DNA c o n t e n t o f sperm of D r o s o p h i l a m e l a n o g a s t e r. Chromosoma ( B e r l . ) 33:1-18. R u f f n e r , J . A. 1980. C l i m a t e s of t h e S t a t e s , N a t i o n a l Oceanic and Atmospheric A d m i n i s t r a t i o n n a r r a t i v e summaries, t a b l e s , and maps f o r each s t a t e w i t h overview o f s t a t e c l i m a t o l o g i s t programs. 2nd ed. V o l . 2. Gale R e s e a r c h , D e t r o i t ( M i . ) . S c h u l t z , L. P. 1929a. D e s c r i p t i o n of a new type o f mud-minnow from western Washington, w i t h notes on r e l a t e d s p e c i e s . Univ. Wash. P u b l . F i s h 2:73-81. S c h u l t z , L. P. 1929b. The d i s c o v e r y of a remarkable t y p e of mud-minnow i n western Washington. J . Pan-Pac. Res. I n s t . 4:12-16. S c o t t , W. B. and E. J . Crossman. 1973. Freshwater B u l l . F i s h . Res. Board Can. 184: 966 pp.  f i s h e s o f Canada.  S e z a k i , K. and H. K o b a y a s i . 1978. Comparison of e r y t h r o c y t e s i z e between d i p l o i d and t e t r a p l o i d i n spinous l o a c h , C o b i t i s biwae. B u l l . Jpn. Soc. S c i . F i s h 44:851-854. S e z a k i , K., H. K o b a y a s i , and M. Nakamura. 1977. Size of erythrocytes i n the d i p l o i d and t r i p i o i d specimens o f C a r a s s i u s a u r a t u s 1 a n g s d o r f i . Jpn. J . I c h t h y o l . 24:135-140. S h a p i r o , H. S. 1976. D e o x y r i b o n u c l e i c a c i d c o n t e n t per c e l l of v a r i o u s organisms. Pages 284-311 j_n_ G. E. Fasman, ed. Handbook o f B i o c h e m i s t r y and M o l e c u l a r B i o l o g y . 3rd ed. Nucleic acids. Vol. I I . CRC P r e s s , C l e v e l a n d (Oh.). Sharma, A. K. and A. Sharma. 1972. Chromosome Techniques. p r a c t i c e . U n i v e r s i t y Park P r e s s , B a l t i m o r e (Md.)  Theory  and  Sherwood, S. W. and J . L. P a t t o n . 1982. Genome e v o l u t i o n i n pocket gophers (genus Thomomys). I I . V a r i a t i o n i n c e l l u l a r DNA c o n t e n t . Chromosoma ( B e r l . ) 85:163-179. S o k a l , R. R. and F. J . R o h l f . 1969. Biometry. The p r i n c i p l e s and p r a c t i c e of s t a t i s t i c s i n b i o l o g i c a l r e s e a r c h . W. H. Freeman, San Francisco (Ca.). S t e v e n s o n , M. M. Cyprinodon.  1981. Copeia  Karyomorphology of s e v e r a l s p e c i e s o f 1981: 494-498.  Subrahmanyan, K. 1969. A k a r y o t y p i c study of the e s t u a r i n e f i s h Boleopthalmus b o d d a r e t i ( P a l l a s ) w i t h c a l c i u m t r e a t m e n t . Curr. S c i . ( B a n g a l o r e ) 38:437-439.  114  Swanson, C. P., T. Merz and W. J . Young. 1981. Cytogenetics. The chromosome i n d i v i s i o n , i n h e r i t a n c e and e v o l u t i o n . 2nd ed. P r e n t i c e - H a l l , Englewood C l i f f s ( N . J . ) . Thompson, K. W. Cichlidae.  1979. Cytotaxonomy o f 41 s p e c i e s of n e o t r o p i c a l Copeia 1979:679-691.  T h o r g a a r d , G. H. 1978. Chromosome rearrangements and sex chromosomes i n t h e rainbow t r o u t and sockeye salmon. Ph.D. d i s s e r t a t i o n , U n i v e r s i t y of Washington, S e a t t l e . Trautman, M. B. 1981. Columbus (Oh.).  The  F i s h e s of Ohio.  T u r n e r , B. J . and R. K. L i u . c o m p a t i b i l i t y i n t h e New 1977:259-269.  Ohio S t a t e U n i v e r s i t y P r e s s ,  1977. Extensive i n t e r s p e c i f i c genetic World k i l l i f i s h genus C y p r i n o d o n . Copeia  Vahs, W. 1973. Die Bedeutung der H y d r o l y s e - A r t i n der F e u l g e n C y t o p h o t o m e t r i e von Kernen m i t u n t e r s c h i e d l i c h e n P l o i d i e g r a d e n . H i s t o c h e m i e 33:341-348. V e n d r e l y , R. 1953. A r g i n i n e and d e o x y r i b o n u c l e i c a c i d content of e r y t h r o c y t e n u c l e i and sperms of some s p e c i e s of f i s h e s . Nature (London) 172:30-31. V e n d r e l y , R. and C. V e n d r e l y . 1956. The r e s u l t s of cytophotometry i n the study of t h e d e o x y r i b o n u c l e i c a c i d (DNA) c o n t e n t of t h e nucleus. I n t . Rev. C y t o l . 5:171-197. W h i t e , M. J . D. 1973. Animal C y t o l o g y Cambridge U n i v e r s i t y , London. W h i t e , M. J . D. 1978. F r a n c i s c o (Ca.).  and  Modes o f S p e c i a t i o n .  Evolution. W.  3rd  ed.  H. Freeman, San  W i l s o n , A. C , G. L. Bush, S. M. Case and M.-C. King. 1975. Social s t r u c t u r i n g of mammalian p o p u l a t i o n s and r a t e of chromosomal e v o l u t i o n . P r o c . Nat. Acad. S c i . USA 72:5061-5065. Wydoski, R. S. and R. R. Whitney. 1979. I n l a n d F i s h e s of Washington. U n i v e r s i t y of Washington P r e s s , S e a t t l e (Wa.). W i l l i a m s , G. C. 1975. Princeton (N.J.).  Sex and  Evolution.  Princeton U n i v e r s i t y Press,  W o l f , K. 1963. P h y s i o l o g i c a l s a l i n e s f o r fresh-water t e l e o s t s . P r o g r e s s i v e F i s h - c u l t u r i s t 25:135-140.  115  W o l f e , J . A. 1981. A c h r o n o l o g i c framework f o r C e n o z o i c m e g a f o s s i l f l o r a s o f n o r t h w e s t e r n North America and i t s r e l a t i o n t o marine geochronology. Pages 39-47 j_n J . M. A r m e n t r o u t , ed. Pacific Northwest Cenozoic B i o s t r a t i g r a p h y . G e o l . Soc. Am. Spec. Pap. No. 184. Zar,  J . H. 1974. B i o s t a t i s t i c a l Analysis. C l i f f s , (N.J.).  P r e n t i c e - H a l l , Englewood  Zenzes, M. T., and I . V o i c u l e s c u . 1976. C-banding p a t t e r n s i n Sal mo t r u t t a , a s p e c i e s of t e t r a p l o i d o r i g i n . G e n e t i c a 45:531-536.  

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