Comparative genetics of Montana and arctic grayling, Thymallus arcticus by Jeremiah Cornelius Lynch A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in Zoology Montana State University © Copyright by Jeremiah Cornelius Lynch (1977) Abstract: An investigation was made of the biochemical genetic variation within and among four populations of the arctic grayling, Thymallus articus. Two populations surveyed were representative of the form found in the main range of the species, northern Canada and Alaska, and two populations were representative of the disjunct Montana form of Thymallus articus. Estimates of these parameters were obtained from a starch gel electrophoretic survey of thirty-five enzyme loci and protein loci. The percent polymorphic loci (12.5 percent) and average heterozygosity (2.7-3.I percent) are intermediate in the range estimated for salmonid species and may reflect the limited habitat diversity of grayling compared with other salmonid species. No relationship between genetic variability and enzyme function was identified for this species. Both a rapidly evolving set and a slowly evolving set of proteins appeared to be present. Comparisons among the four populations were based on allelic protein variation at eight loci. Results of genetic similarity and genetic distance calculations indicate that genetic divergence has taken place between the arctic form and Montana form of T. arcticus, which may warrent subspecific status for the two forms.  STATEMENT OF PERMISSION TO COPY In p re sen t ing t h i s t h e s i s in p a r t i a l f u l f i l lm e n t o f the requirements f o r an advanced degree a t Montana S t a t e U n iv e r s i ty , I agree t h a t the Library s h a l l make i t f r e e l y a v a i l a b l e f o r in spec ­ t i o n . I f u r t h e r agree t h a t permission fo r ex ten s ive copying of t h i s t h e s i s f o r s cho la r ly purposes may be g ran ted by ny major p ro f e s so r , o r , in h is absence, by the D i re c to r o f L i b r a r i e s . I t i s understood t h a t any copying o r pub l i c a t ion o f t h i s t h e s i s f o r f i n an c ia l gain sh a l l not be allowed wi thout my w r i t t e n permiss ion . COMPARATIVE GENETICS OF MONTANA AND ARCTIC GRAYLING, THYMALLUS ARCTICUS by Jeremiah C. Lynch A t h e s i s submitted in p a r t i a l f u l f i l lm e n t o f the requirements f o r the degree of MASTER OF SCIENCE in Zoology Approved: i ^ = __________ Chairperson, Gj^duate Committee 'ad, Major Department Graduate Bean MONTANA,STATE UNIVERSITY Bozeman, Montana September, 1977 i i i ACKNOWLEDGMENTS I would l i k e to express my s in ce re g r a t i t u d e to my major p ro f e s ­ so r , Dr. E rnes t R. Vyse, f o r h i s guidance , a s s i s t a n c e and continued support throughout t h i s s tudy. Special thanks are extended to John D. Varley (Yellowstone Park F ishe r ie s Management) f o r h is i n t e r e s t in the s tudy and a s s i s t a n c e in ob ta in ing samples; Dr. David G. Cameron f o r h i s a s s i s t a n c e through out t h i s s tudy and c on s t r u c t i v e review o f t h i s manuscr ip t ; Dr. Fred W. Al lendorf (Un ive rs i ty o f Montana) f o r the use o f h i s computer program fo r a n a ly s i s o f the d a ta ; and Dr. Calvin Kaya fo r h i s review o f t h i s manuscript . F in a l l y , I would l i k e to thank my w i fe , T e r i , f o r the spec ia l support she has given me throughout t h i s s tudy. TABLE OF CONTENTS Page VITA . ........................................................................................... i1 ACKNOWLEDGMENTS ............................................................................................ i i i LIST OF TABLES............................................................................................... vi LIST OF FIGURES........................................... v i i ABSTRACT........................................... ix INTRODUCTION ............................................................. I D i s t r i b u t io n ........................................................................................ I Taxonomy ............................................ E lec t ropho re s i s . . . . . . . . Va r ia t ion in Natural Popula tions O b j e c t i v e s ............................................................................................ 13 MATERIALS AND METHODS.......................... 19 Sampling o f Populations : .............................................................. 19 Sample P r e p a r a t i o n .......................... 22 E l e c t r o p h o r e s i s . ...................................................................................... 22 Qua l i t a t i v e Analysis ....................................................................... 24 Nomenclature ........................................................................................ 29 RESULTS.......................... 31 E l e c t ropho re t i c Phenotypes o f Monomorphic P ro te in s . . 31 Lac ta te dehydrogenase ............................... 31 Malate dehydrogenase ....................................... 35 Glutamat e - o x a l o ace ta t e transaminase .............................. 38 Alcohol dehydrogenase ................................................ . . . 42 Xanthine dehydrogenase ......................................................... 42 So rb i to l dehydrogenase ......................................................... 43 I s o c i t r a t e dehydrogenase ..................................................... 44 Alpha-glycerophosphate dehydrogenase . . . . . . . 45 E s t e r a s e ................................... 46 H e x o k i n a s e .................................................................................... 47 LO I''- O O TABLE OF CONTENTS (Continued) Page E lec t ropho re t i c Phenotypes o f Polymorphic P ro te in s . . 47 Tetrazolium oxidase .................................................................. 47 Phosphoglucomutase .................................................................. 50 I s o c i t r a t e dehydrogenase .............................. 52 T r a n s f e r r i n .................................................................................... 56 Glucose and hexose 6-phosphate dehydrogenase . . . 59 Malic e n z y m e .......................... 68 Serum p ro te in s .......................................................................... 71 Quan t i t a t iv e Analysis o f Genetic V a r i a b i l i t y .................. 80 DISCUSSION......................................................... .... . ; ........................... 89 Genetic V a r i a b i l i t y o f Thymallus cwctieus ....................... 89 Genetic Divergence Between Popula tions o f Thymallus a v o t io u s ................................................ ' .............................................. 97 Taxonomic Considera t ions . ............................................................. 107 APPENDIX............................................................................. HO Buffer Systems .................................................................. .... . . . I l l S ta in ing Procedures .............................................................................. 113 V LITERATURE CITED 117 /vi LIST OF TABLES Table Page 1. Co l lec t ion data f o r the fou r popula t ions o f Thymallus avotious ..................................................... .... 21 2. P ro te in s surveyed, t i s s u e s examined, and bu f f e r systems employed in e l e c t r o p h o r e t i c a n a ly s i s o f Thymallus arctleus . ................................... . . . . . . . 27 3. A l l e l e f r equencies and degree o f he te rozygos i ty in 35 loc i examined in fou r popula t ions o f Thymallus oFctious ...................................................................................... 81 4. Correspondence o f observed genotype f requenc ies to those expected on the ba s i s o f Hardy-Weinberg equ i l ib r ium f o r the polymorphic loc i o f Thymallus a v o t i o u s ..............................................................................................................................................................................................................................................................................86 5. Estimates o f gene t ic v a r i a b i l i t y in Thymallus avotious ...................................................................................................................................................... 88 6. Amount o f polymorphism and the degree o f h e t e r o ­ zygos i ty in some f i s h spec ies ........................................ .... . 91 7. Indices o f s im i l a r i t y and gene t ic d i s t a nce f o r . four popula t ions o f Thymallus avotious ........................... 99 8. Genetic s im i l a r i t i e s between popula t ions a t d i f f e r e n t s tages o f evo lu t iona ry divergence in severa l groups o f f i s h ..................................................... .... . 102 vi i LIST OF FIGURES Figure Page 1. Map o f Alaska and western Canada showing g ray l ing d i s t r i b u t i o n ............................................................................... . 2 2. D i s t r i b u t i o n o f indigenous g ray l ing in Montana . . . 4 3. Lac ta te dehydrogenase (LDH) ..................................................... 34 4. Tissue d i s t r i b u t i o n o f mala te dehydrogenase (MDH) from the same f i s h .............................................................................37 5. Glu tamate-oxaloace ta t e t ransaminase (GOT) t i s s u e d i s t r i b u t i o n ............................................................. 40 6. Tetrazolium oxidase (TO) polymorphism ............................... 49 7. Phosphoglucomutase (PGM) ......................................................... 51 8. Phosphoglucomutase (PGM) polymorphism ............................... 53 9. I s o c i t r a t e dehydrogenase (IDH) ............................................ 55 10. T r an s f e r r in (Tfn) polymorphism . ..............................................58 11. Glucose-6-phosphate dehydrogenase (G6PD) and hexose- 6-phosphate dehydrogenase (H6PD) express ion in e ry th rocy te s and eye t i s s u e ...........................................................63 ' 12. Glucose-6-phosphate dehydrogenase-3 (G6PD-3) . . . . 64 13. Hexose-6-phosphate dehydrogenase (H6PD) po ly ­ morphism .................................................................................................. 65 14. Malic enzyme (ME) ........................................................................... 70 15. Electropherograms of serum p ro t e in s .................................. 74 16. Diagrammatic r e p r e s en ta t i o n o f e l e c t r o p h o r e t i c p a t t e rn o f serum p ro te in s o f Thymallus avc tious . . . 76 v i i i LIST OF FIGURES (Continued) Figure Page 17. The twelve observed phenotypic p a t t e r n s of e l ectropherograms o f g ray l ing serum p ro te in s in Zone 5 ............................................................................................ 77 18. Dendrogram fo r four popula t ions o f T. aroti-cus . . . 109 ix ABSTRACT An in v e s t i g a t i o n was made o f the biochemical g ene t i c v a r i a t i o n wi th in and among fou r popu la t ions o f the a r c t i c g r a y l in g , Tkymallus avo tious. Two popula t ions surveyed were r e p r e s e n t a t i v e o f th e form found in the main range o f the s p e c i e s , no r the rn Canada and Alaska, and two popu la t ions were r e p r e s e n t a t i v e o f the d i s j u n c t Montana form o f Tkymallus avo tiaus. Estimates o f th e se parameters were ob ta ined from a s t a r ch gel e l e c t r o p h o r e t i c survey o f t h i r t y - f i v e enzyme loci and p ro te in l o c i . The pe rcen t polymorphic lo c i (12.5 p e rc en t ) and average h e te rozygos i ty ( 2 . 7 - 3 . I pe rcen t ) a re in te rmed ia te in the range e s t ima ted fo r salmonid spec ie s and may r e f l e c t the l im i t ed h a b i t a t d i v e r s i t y o f g ray l ing compared with o th e r salmonid s p ec ie s . No r e l a t i o n s h i p between g ene t i c v a r i a b i l i t y and enzyme func t ion was i d e n t i f i e d f o r t h i s sp ec ie s . Both a r a p id ly evolv ing s e t and a slowly evolv ing s e t of p ro t e in s appeared t o be p re s en t . Comparisons among the fou r popula t ions were based on a l l e l i c p ro te in v a r i a t i o n a t e ig h t l o c i . Result s o f g ene t i c s im i l a r i t y and gene t ic d i s t a n ce c a l c u l a t i o n s i n d i c a t e t h a t g ene t i c divergence has taken p lace between the a r c t i c form and Montana form o f T. apc tiou s, which may warrent s ub sp e c i f i c s t a t u s f o r the two forms. i INTRODUCTION D is t r ib u t ion Thymallus a vo tieu s , the a r c t i c g r a y l in g , i s a f r e shwa te r f i s h which inh ab i t s cold o r a r c t i c reg ions . The n a t iv e range o f the spec ie s is hoi a r c t i c , occur r ing in nor the rn d ra inages o f North America and Euras ia . In Euras ia , i t i s found from the Kara and Ob R ive r s , in the western U.S.S.R. to th e e a s t e rn S ibe r ian Coast ( inc lud ing a l l streams d ra in ing in to the Bering Sea, and the Penzhina River d ra in ing in to the sea o f Okhotosk), south to nor the rn Mongolia and the Yalu River (Walters 1955, S co t t and Crossman 1973). In Canada and Alaska, T. a ro tious occurs from V a n s i t t a r t I s land o f f the Melv i l le Pen insu la ; south along the west coas t o f Hudson Bay to the Owl River , Manitoba; west throughout t h e ^Northwest and Yukon T e r r i t o r i e s to the Bering Sea dra inages in Alaska; south in Saska tche­ wan to Reindeer Lake but absen t in most o f the Churchil l R iver ; south to Central A lbe r ta ; in nor the rn B r i t i s h Columbia from the Pease and S t ik ine River north (Walters 1955, Slas tenenko 1950, S co t t and Crossman 1973). Figure I shows the d i s t r i b u t i o n o f Thymallus avc tious in Canada and Alaska. In the contiguous United S t a t e s Thymallus a ro tious was indigenous in Michigan and Montana. I s o l a t e d popula t ions were p re s en t in Michigan in the upper p a r t o f the Lower Pen insu la , and in the O t t e r 3River o f the Upper Peninsula (Hubbs and Lagle r 1949). The Michigan form, however, has been e x t i n c t s ince 1936 (Sco t t and Crossman 1973, U.S. Dept, o f I n t e r i o r 1966). Another popula t ion was found in Montana in the headwaters of the Missouri River above the Great F a l l s (Henshall 1906). This southward extens ion and the subsequent e s t a b ­ lishment o f these two popula t ions was ev id en t ly the r e s u l t o f g l a c i a l ac t ion . In Montana, the o r i g i n a l range as desc r ibed by Henshall (1906) has been g r e a t l y reduced. The dec l ine o f the spec ie s has been r epo r ­ ted and rea f f i rmed by var ious i n v e s t i g a t o r s (Kelly 1931, Brown 1943, Nelson 1954, 1956). The p re sen t d i s t r i b u t i o n in n a t iv e r i v e r s and streams i s descr ibed by Brown (1971) with t h e s ta temen t t h a t "a few are found in the Sun, Big Hole, Red Rock, and Madison R i v e r s . ". Gray­ l ing a re e n t i r e l y absent from the Missouri R iver , the G a l l a t i n River and the main stem o f the J e f f e r son River. Two small remnant popula­ t io n s remain in the J e f f e r son t r i b u t a r i e s : one in the Big Hole River \ and the o th e r in the Red Rock Lakes area (Nelson 1954). The range o f indigenous popula t ions o f Thymallus a^o tious in Montana i s shown in Figure 2. There has been widespread t r a n s p l a n t i n g o f Canadian s tocks in to Montana popula t ions (McPhail and Lindsey 1971) and ha tchery p l a n t in g from a s in g l e source ( the Red Rock Lakes) i n to a l l but one o f the 4Great Falls Columbia Riirer Drainage Helena p IFnrk s Y el low stone National Park Idaho Figure 2, D is tr ibu tion of Indigenous grayling in M on ta n a . 5na tu ra l popula t ions (Kelly 1931). At t h i s t ime reproducing g ray l ing popula t ions are known t o e x i s t in 39 lakes and 14 s treams in western Montana on both s ides o f the Continenta l Divide (Hblten 1971). The only indigenous Montana popula t ion known not to be contaminated by p lan t ing s i s the Red Rock Lakes popula tion (Nelson 1954). In Yellowstone National Park the g ray l ing occurred n a t u r a l l y in the Madison River system and the G a l l a t i n River . I t i s no longer p re sen t in the G a l l a t in River and i s r a r e in the Madison River (Dean and Varley 1974). T ransp lan t ing o f g ray l ing to lakes in Yellowstone National Park has been desc r ibed by Kruse (1959). Successfu l s e l f - ' propagating popula t ions were e s t a b l i s h e d in Grebe Lake, Wolf Lake, and Ice Lake, above the V i rg in i a Cascades on the Gibbon R iver , and Cascade Lake in the Yellowstone dra inage . Taxonomy The g ray l ings a re s o f t rayed t e l e o s t f i s h belonging to the o rde r Isospondyli , suborder Salmonoidei. The ir f u r t h e r taxonomic c l a s s i f i c a ­ t ion has been one f raugh t with confus ion. The genus Thymallus was separa ted from the genus Salmo (Curvie r 1829), bu t t h e i r family c l a s s i f i c a t i o n was debated f o r some time by taxonomists and remains unresolved today (Sco t t and Crossman 1973). Some au thors have ass igned a l l th re e geographic groups (A rc t i c , Asian, and Montana-Mic h igan) to the Salmonidae (Boulenger 1895, Regean 1914) while o the r s (Jordan and 6Everman 1896, Berg 1940, 1955) have placed the g ray l ings in a s ep a ra te family , th e Thymmalli d a e . The most r e c en t c l a s s i f i c a t i o n , based on o s teo log ica l c h a r a c t e r i s t i c s (Norden 1961), p laces th e g ray l ing in the sub-family ThymaU l n a e , o f the family Salmonldae. Four spec ie s a re recognized: T. b rev iro stv is (Mongolia), T. thymallus (Europe) , T. nigvesoens (Lake Kosogol, Mongolia) and T. arotious (e a s te rn S ib e r i a and North America). The s t a t u s o f the var ious forms o f araticus has been debated fo r some time. For decades i t was considered t h a t the North American g ray l ings con s i s t e d o f th r e e s p e c i e s : TH sign ifev (Richardson 1823) found in nor the rn Canada and Alaska, T. tr ic o lo r (Cope 1865) found in Michigan, and T. montanus (Milner 1873) found in Montana. This c l a s s i f i c a t i o n was p r i n c i p a l l y based on geographic i s o l a t i o n , and severa l morphological c h a r a c t e r i s t i c s ( s i z e and shape o f dorsa l f i n , max i l la ry l e ng th , and c o lo r v a r i a t i o n ) . More r e c e n t l y , T. s ign ife r has been cons idered conspec i f ic with TH arotious ( P a l l a s ) , and the o th e r American forms r e l ega ted sub sp e c i f i c s t a t u s (Walters 1955). Walters showed t h a t the Canadian and Alaskan form was i d e n t i c a l with two A s i a t i c forms (y. a. p a lla s i and T. a. gruberi natio mertensi) and suggested t h a t they be des igna ted as Thymallus arotious s ign ife r (Richardson 1823). Walters (1955) f u r t h e r recognized the Montana- Michigan form as ano ther subspec ies tr ico lo r .. The v a l i d i t y o f the 7North American subspecies has not been adequate ly demonst rated (Sco t t and Crossman 1973, Norden 1961). At the p re sen t time no subspec ies should be recognized w i th in T. arcticus un t i l f u r t h e r evidence warrants such d i s t i n c t i o n (McPhail and Lindsey 1971). E lec t rophore s is P ro te in s a re ampholytes and, t h e r e f o r e , may ca r ry a n e t nega t ive or p o s i t i v e charge. The n e t charge depends on the i o n i z a t io n o f I ) f r ee carboxyl groups (COOK™) o f glutamic a c id and a s p a r t i c ac id res idues and 2) f r e e amino groups (NH^+) o f ly s in e and a rg in in e (and to a l e s s e r e x t e n t h i s t i d i n e ) . The ne t charge o f the p ro te in depends, on which group predominates. The degree o f i o n i z a t io n depends on the pH of the p ro t e in s o lu t io n . In a bu f f e r o f high pH the a c i d i c groups a re p rog re s s iv e ly n e u t r a l i z ed by the a l k a l i component o f the b u f f e r , thus a llowing the bas ic groups t o predominate. This r e s u l t s in the p ro te in molecule having a n e t nega t ive charge. At a low pH the reverse occu r s , and the p ro te in w i l l have a ne t p o s i t i v e charge. At a c e r t a i n pH, the i s o e l e c t r i c p o in t , the p o s i t i v e and nega t ive charges a re balanced and th e re i s no ne t charge. E lec t ropho re s i s manipula tes the ampholytic behavio r o f p ro te in s by applying an e l e c t r i c a l f i e l d to a s o lu t io n o f p r o t e i n s , s ep a ra t ing them on the b a s i s o f t h e i r ne t charge. I f the pH i s l e s s than the i s o e l e c t r i c po in t o f the p ro t e i n , i t w i l l m ig rate toward the cathode 8and i f the pH i s g r e a t e r than the i s o e l e c t r i c po in t o f the p ro t e i n , i t w i l l mig rate toward the anode. The r a t e o f migra t ion depends on the number o f charges , the molecular s i z e and on the vo l tage app l ied . There fore , by the s e l e c t i o n o f the app rop r i a t e bu f f e r and e l e c t r i c a l c u r r e n t , p ro te in d i f f e r en ce s can be determined. E l e c t ropho re t i c techn iques vary as to the suppor t ing media used. Starch gel was employed in the p re s en t s tudy . Isozyme sep a ra t ion using s t a r ch gel depends not only on n e t ion ic charge , but to a l e s s e r e x t en t on d i f f e r en ce s o f molpcular s i z e . S ta rch gel a c t s as a molecular s i e v e , mechanical ly s ep a ra t ing molecules o f d i f f e r e n t s iz e s by a f f e c t i n g t h e i r r a t e o f migra tion (Smith ies 1955). Var ia t ion in Natural Popula tions The development o f s t a r ch gel e l e c t r o p ho r e s i s (Smithies 1955), and t h e i n t r oduc t ion of simple s t a i n in g techn iques f o r the d e te c t ion o f s p e c i f i c enzyme a c t i v i t y (Hunter and Markert 1957), has allowed the v i s u a l i z a t i o n o f ind iv idua l p r o t e i n s , hence, s i n g l e gene products The combination o f these techn iques provided a means by which h e t e r o ­ genei ty o f p ro t e in s and enzymes could e a s i l y be d e tec ted . This allowed the c h a r a c t e r i z a t i o n a t the molecular level o f the amount o f gene t ic v a r i a b i l i t y in popula t ions and an e s t ima te o f the ex ten t o f gene t ic d ivergence among c lo s e ly r e l a t e d spec ie s (Go t t l i eb 1971). 9Kimura and Crow (1964) hypothesized t h a t the number o f a l l e l e s t h a t can be maintained a t a s in g l e locus in a f i n i t e popu la t ion i s l a rg e . Shaw (1965) poin ted out t h a t isozymes which vary w i th in popu­ l a t i o n s i s the ru l e r a t h e r than the excep t ion . Approximately TOO loc i are known to have e l e c t r o p h o r e t i c v a r i a n t s in popu la t ions o f many organisms, inc lud ing man. Drosophila, a n t s , f i s h , mice , f rogs and many p l a n t spec ies (Go t t l i eb 1971). The amount o f such genic v a r i a t i o n , measured by the p ropor t ion o f polymorphic loc i (common a l l e l e frequency l e s s than or equal to 0 .99) can be determined d i r e c t l y from e l e c t r o p h o r e t i c a n a ly s i s . In th eo ry , a l a rge number o f s t r u c t u r a l l y d i f f e r e n t a l l e l e s may be generated by independent muta tions w i th in the conf ines o f a s ing le gene (Harr is 1976). A c e r t a i n p ropor t ion o f these muta tions can be expected to r e s u l t in a s u b s t i t u t i o n a f f e c t i n g the ne t charge o f the p ro te in . Such a mutation would be r e f l e c t e d in the mob i l i ty o f the p ro te in . The p r o b a b i l i t y t h a t such a mutat ion w i l l occur i s c a l c u l a ­ ted to be 25-30% (Shaw 1965, Nei 1975). This means t h a t a l a rge number o f amino ac id s u b s t i t u t i o n s go undetec ted s ince they do not r e s u l t in a charge change. The e s t im a te s o f gene t i c v a r i a b i l i t y based on e l e c t r o p h o r e t i c a l l y d e t e c t a b l e d i f f e r en c e s may, t h e r e f o r e , be conserva t ive (Harr i s and Hopkinson 1976, Nei 1975). 10 Data from a random sample o f loc i coding f o r p ro t e in s can be ex t r apo la t ed t o e s t imate the amount o f g ene t i c v a r i a b i l i t y in the e n t i r e genome (Lewontin and Hubby 1966). The e s t im a te s o f the amount o f polymorphism in the sample can be used to c a l c u l a t e the p ropo r t ion o f polymorphic loc i and ind iv idua l h e te rozygos i ty in a s p ec ie s . These e s t im a te s can provide a b a s i s f o r comparison between spec ies (U t te r e t a l. 1973) and may a l so be used to compare popula t ions wi th in spec ie s . The level o f gene t ic polymorphism has been e s t imated fo r a v a r i e ty o f sp ec ie s : man - 25 pe rcen t o f 12 loc i were polymorphic (Harr is 1966) and more r e c en t ly man - 31 pe rcen t o f 71 loc i (Harr is and Hopkinson 1972); Mus rmsoulus - 30 pe rcen t polymorphic (Selander e t a l. 1969) and 40 percen t polymorphic (Selander and Yang 1969); Pevomysous polionotus - 23 pe rcen t polymorphic (Selander e t a l. 1971); q u a i l , Cotuvnix ootuvnix - 54 to 58 pe rcen t polymorphic (Baker and Manwell 1967); pheasan t , Phasianus ooldhious - 43 pe rcen t polymorphic (Baker e t a l. 1966); Dvosophila ( v a r i e ty o f sp ec ie s ) - 30 to 67 p e r ­ cen t polymorphic ( Lewontin and Hubby 1966, Prakash e t a l, 1969, Ayala e t a l. 1970, Berger 1970, O'Brien and MacIntyre 1969). In f i s h s p e c i f i c a l l y , e s t ima te s a re : Astynax - 29 to 41 pe rcen t f o r in land popula t ions and 0 to 20 p e rc en t f o r cave dwel lers (Avise and Se lander 1972), he r r ing - 45 percen t polymorphic (Altukhov e t a l. 1972), brook 11 t r o u t , Salvelinus fon tina lis - 38 pe rcen t polymorphic (Wright and Atherton 1970); chum salmon - 11 to 18 pe rcen t polymorphic (Altukhov e t a l. 1972); ro ck f i s h , Sebastes ( v a r i e ty o f sp ec ie s ) - 4 to 8 pe rcen t polymorphic (Johnson e t a l. 1973), P a c i f i c salmon - 8 to 13 pe rcen t (U t te r e t a l . 1 9 7 3 ) rainbow t r o u t - 26 pe rcen t (U t t e r e t a l . 1973). In surveying the l i t e r a t u r e as a whole, approximate ly 30 pe rcen t o f the s t r u c t u r a l gene loc i a re polymorphic (Go t t l i eb 1971; Nei 1975). Heterozygosity v a r ie s cons ide rab ly with locus (King and Wilson 1975, Nei and Roychoudhury 1974, Se lander and Johnson 1973, U t t e r e t a l. 1973). The ex is t ence o f locus dependent r a t e s o f change i s well i l l u s t r a t e d by amino ac id sequencing da ta in d ive r s e organisms (Dickerson 1972), which in d i c a t e s t h a t p ro t e in s evolve a t d i f f e r e n t r a t e s . Gene lo c i a re polymorphic when a l l e l e s u b s t i t u t i o n i s in t r a n s i ­ t i o n , when ba lanc ing s e l e c t i o n s t a b i l i z e s f r equ en c i e s , or when a mutant a l l e l e becomes f r equen t by chance. Since each locus may under- I go a l l e l e s u b s t i t u t i o n independent ly , a high degree o f i n t e r l o c u s v a r i a t i o n in he te rozygos i ty may r e s u l t . I n t e r lo cu s v a r i a t i o n may a l so be produced i f the mutation r a t e o r the type and i n t e n s i t y o f na tu ra l s e l e c t i o n v a r i e s among loc i (Nei 1975). That loc i v a r i a t i o n and, hence, p ro te in h e te rogene i ty i s p re sen t in a wide v a r i e t y of v e r t eb ra te spec ie s was shown by Se lander and Johnson (1973). 12 In te r lo cu s v a r i a t i o n has a l so been found in many spec ie s o f f i s h (U t te r e t a t . 1973). This i n t e r l o c u s v a r i a t i o n can be sepa ra ted in to two groups, a r ap id ly evolv ing s e t and a slowly evolv ing s e t . The r a p id ly evolv ing s e t , inc lud ing plasma p ro te in s and e s t e r a s e s , accumulates e l e c t r o - p h o r e t i c a l l y d e t e c t ! ble s u b s t i t u t i o n s a t a r a t e t e n fo ld g r e a t e r than the slower s e t , which inc ludes enzymes involved in metabo l ic pathways (Sar ich 1977). Cor re la t ion s have been proposed between enzyme func t ion and he te rozygos i ty (G i l l i s p i e and Kojima 1968, Kojima e t a t . 1970, Johnson 1971, 1974, Powell 1975), and more r e c e n t l y a r e l a t i o n ­ sh ip between he te rozygos i ty and qua te rnary s t r u c t u r e has been proposed (Ward 1977). The bas is f o r the d i f f e r en c e in h e te ro zygos i ty between loc i i s not adequate ly r e so lv ed , but e s t im a te s o f average h e t e r o ­ zygosi ty o r g ene t i c d i s t a nce would be overes t imated o r underest imated i f the p ro t e in s chosen did not inc lude an adequate mixture o f both s e t s . In the p re s en t s tudy a l a rg e number o f loc i have been examined with no p re fe rence given to e i t h e r the r a p id ly evolv ing or the slower evolving s e t o f p r o t e in s . Nei and Roychoudhury ( 1974) have sugges ted t h a t to e s t im a te the average h e te rozygos i ty per lo cus , a la rge number o f loc i r a t h e r than a la rge number o f ind iv idua l s should be used. Avise and Ayala (1975) s t a t e t h a t with r e sp ec t to gene t i c s im i l a r i t y , the va r iance about 13 i nd iv idua ls is. small r e l a t i v e to the va r iance about l o c i ; t h e r e f o r e , the p re c i s ion o f these e s t im a te s i s much more dependent on the number of loc i than on the numbers o f ind iv idua l s sampled. I f the i n t e n t i s to p r e d i c t Hardy-Weinberg equ i l ib r ium as well as e s t im a te average h e te ro zygos i ty , a r e l a t i v e l y l a rge number o f i n d iv id u a l s should be examined f o r each polymorphic locus (Nei 1975). I f both o f these es t imated a re w i th in the scope o f a s tudy , a l a rge number o f i n d iv id u ­ a l s should be screened a t a l a rge number o f lo c i with the hope o f minimizing any b ia s . A g r e a t deal o f da ta has been c o l l e c t e d as to the gene t i c v a r i a ­ b i l i t y o f f i s h p ro te in s (deLigny 1969, Kirpichnikov 1973). P ro te in he te rogene i ty appears to be p re s en t in nea r ly every spec ie s s tud ied . A r e l i a b l e s e t o f p ro te in s which have been s tud ied in o th e r f i s h s p ec ie s , and repo r ted in the l i t e r a t u r e , were chosen f o r a n a ly s i s so t h a t r e l i a b l e e s t im a te s o f average h e te rozygos i ty and Hardy-Weinberg f requencies could be c a l c u l a t e d . In a d d i t i o n , comparisons can be made to the publi shed r e s u l t s f o r o th e r sp ec ie s . The a v a i l a b i l i t y o f s u b s t r a t e f o r s t a i n i n g and the c l a r i t y o f r e s o lu t i o n were the f i n a l f a c to r s in dete rmin ing what p ro te in s were inc luded. f Object ives The North American forms o f Thymallus avctieus have been i s o l a t e d s ince before th e l a s t Wisconsin g l a c i a t i o n (Vincent 1962). I s o l a t ed 14 popula t ions were e s t a b l i s h e d in favorab le h a b i t a t s south o f the main range o f Thymallus ccrotious. For popula t ions to become g e n e t i c a l l y d i f f e r e n t i a t e d , e v o lu t i o n i s t s be l ieve they must be completely i s o l a t e d from one ano ther . This i s o l a t i o n may occur geog raph ica l ly o r r e p roduc t iv e Iy (Dobzhansky 1951 , 1970). Complete i s o l a t i o n has occurred in th e case o f Thymallus arotious, between the a l l o p a t r i c popu la t ion s , which a t one t ime shared the same gene pool , but have s ince become i s o l a t e d from one ano ther . The re fo re , an oppor tun i ty f o r e vo lu t iona ry divergence o f th e var ious s tocks has been p re s en t . An es t ima t ion o f the amount o f divergence which has taken p lace could be obta ined by a survey o f e l e c t r o p h o r e t i c d i f f e r e n c e s (d iscussed p rev iou s ly ) . However, the comparison o f the th r e e american forms o f T. avetious meets with some d i f f i c u l t y . The Michigan form has been e x t i n c t s ince 1936 (Sco t t and Crossman 1973, U.S. Dept, o f the I n t e r i o r 1966) and thus i t s d ivergence from the o th e r two cannot be determined. The Montana form as desc r ibed by HenshalI (1906) d e a l t almost e x c lu s iv e ly in r i v e r s and s t reams , hence i t was an ad f lu v i a l form (s tream dwel l ing -s t ream spawning) and only s econda r i ly a l a cu s ­ t r i n e form (lake dwel l ing-s t ream spawning). From a g ene t i c and taxonomic p o in t o f view, the s tudy o f the a d f lu v i a l form may be impossible due to the reduced numbers (Brown 1971), the contaminat ion o f independent popula t ions by the i n t r oduc t ion o f Canadian s tocks 15 (MePhaiI and Lindsey 1971), and the un ive rsa l p l an t ing o f l a c u s t r i n e forms from a s i n g l e donor (Red Rock Lakes) i n to d i s c r e t e a d f lu v i a l popu la t ions . The Red Rock Lakes popula t ion i s a pure deme o f the Montana form o f Thymallus Ca1O tiousi with no t r a n s p l a n t s o f any o th e r s tocks having taken place (Nelson 1954). The Red Rock Lakes popula­ t i o n , a l though known to be n a t i v e , i s a r e s t r i c t e d headwaters popula­ t ion and may have been g e n e t i c a l l y d i s t i n c t in i t s own r i g h t . The use of i t as a donor s tock in widespread p l a n t in g may have d i l u t e d the enzoo t ic popula t ions in r i v e r s and streams throughout the n a t ive range. The v a l i d i t y of a gene t i c ba s i s f o r the ad f lu v i a l and l a cu s ­ t r i n e behavioral d i f f e r en c e s has not been s t u d i e d , however, the inna te gene t ic con t ro l o f migration in o th e r salmonid spec ie s has been suggested (Raleigh 1967, Brannon 1967, Northcote 1969, Raleigh and Chapman 1971). I f th e re a re d i s t i n c t g e n e t i c a l l y c o n t r o l l e d behav­ io ra l d i f f e r en c e s between the two forms, i t may be sugges ted t h a t the \ l a c u s t r i n e form, h i s t o r i c a l l y r a r e in the Montana range o f the s p e c i e s , i s now widespread while the h i s t o r i c a l l y common a d f lu v i a l type i s th rea tened with e x t i n c t i o n . In the p re sen t s tudy , the Grebe and Wolf Lakes popu la t ion s , which were e s t a b l i s h e d by t r a n s p l a n t i n g from a s tock der ived from the Madison River system (Kelly 1931, Kruse 1959), were used to r ep re sen t the Montana form. Grayling were not na t ive to e i t h e r o f th e se l a k e s . 16 but both were stocked with g ray l ing der ived only from the Madison R iv e r system. Eggs taken from g ray l ing na t ive to Meadow Creek in the Madison River dra inage were rea red in the S ta te Fish and Game's Anaconda ha tchery and the progeny were p lan ted in Georgetown Lake (Kelly 1931). Subsequent to t h i s p l a n t i n g , eggs taken from the se Georgetown g ray l ing were t r a n sp l an t ed to these lakes in Yellowstone Park (Kruse 1958). The da ta obta ined from the se demes. i s , t h e r e f o r e , presumed to be of an ad f lu v i a l form which have s ince become adapted to a l a c u s t r i n e ex i s t ence . The da ta ob ta ined can be used to e s t ima te the amount of divergence which has taken place between the A rc t i c form and the Montana forms o f Thymallus arotious. This e s t ima te o f the amount o f gene t ic divergence can be based on the g ene t i c c h a r a c t e r s o f the popula t ions ob ta ined through e l e c t r o p h o r e t i c da ta . The use o f the Madison River popula tion i t s e l f i s a p r a c t i c a l im po s s i b i l i t y due to the diminished numbers o f ind iv idua l s in t h i s d e c l in ing popu la t ion (Dean and Varley 1974). In add i t io n to the comparison o f the A rc t ic form and the Montana form, the s tudy can a l so determine i f g ene t i c d i f f e r e n t i a t i o n has occurred w i th in two Montana popu la t ion s , the Grebe Lake and Wolf Lake popu la t ions . Salmonids have a tendency to evolve g e n e t i c a l l y d i s c r e t e , e c o lo g i c a l ly s p e c i a l i z e d popula t ions with d i f f e r e n t i a t i o n based on l i f e h i s t o r y c h a r ac te r s such as t ime and place o f spawning 17 (Behnke 1972). As p rev ious ly mentioned, inna te g ene t i c con t ro l o f migra tion h ab i t s in salmonids has been sugges ted . The s t rong homing behavior o f most salmonids i s an important f a c t o r in t h e genera t ion and maintenance o f t h i s g ene t i c d i v e r s i t y (A l lendorf e t a l . 1971). Such appears t o be the case with these p o pu l a t i o n s , the Wolf Lake popula tion sampled was an o u t l e t spawning popu la t ion while the Grebe Lake popula t ion sampled was an i n l e t spawning popu la t ion . Thus, the Grebe and Wolf Lakes popula t ions have p a r t i a l Iy e s t a b l i s h e d e t h o lo g i - cal rep roduc t ive i s o l a t i o n , which may be the f i r s t s t ep in gene t i c i s o l a t i o n . The amount o f g ene t i c divergence between th e se two popu­ l a t i o n s thus gives an e s t ima te o f the amount o f g ene t i c change, a t the s t r u c t u r a l gene l e v e l , t h a t has accompanied t h i s even t . I f the magnitude o f the divergence between these popu la t ions and the Canadian popula t ions i s l a r g e , the evo lu t iona ry p o s i t io n o f th e se forms could be c l a r i f i e d . The phenotypic f r equencies revea led by p a t t e r n s o f p ro t e in s on e l e c t r o p h o r e t i c g e l s , can be i n t e r p r e t e d in terms o f genotypic f r e ­ quencies and the popula tion a l l e l i c f r e q u e n c i e s , which a re the parameters o f e vo lu t iona ry g ene t i c s . With these two parameters of the dernes known, the i n l e t and o u t l e t spawning popu la t ions can be compared with one ano ther and with the A rc t ic form with regard to the unique p ropor t ion o f the genome t h a t d i s t i n g u i s h e s these 18 popu la t ions , and the level o f he te rozygos i ty under the d i f f e r e n t environments. Both o f these a re important p r o p e r t i e s o f d ive rg ing gene t ic systems. E le c t ropho re t i c techn iques provide cons ide rab le informat ion to help e l u c i d a t e evo lu t iona ry r e l a t i o n s h i p s among, c l o s e ly r e l a t e d spec ies (Avise 1974). Biochemical gene t i c v a r i a t i o n among c lo s e ly r e l a t e d popu la t ions can be used t o examine sy s tema t ic r e l a t i o n s h i p s (Nei 1975, Sar ich 1977). The use o f such da ta to determine taxonomic s t a t u s has been su c c e s s fu l ly app l ied to salmonid spec ie s (Payne e t d l. 1971, Nyman 1972, U t te r e t a t. 1973, Re in i tz 1974). The da ta ob ta ined in the p re sen t s tudy w i l l c o n t r ib u t e to the da ta needed to help c l a r i f y the confused taxonomic p o s i t io n t h a t p r e s en t ly e x i s t s through the subspecies c l a s s i f i c a t i o n o f Thymallus aratious. The r e s u l t s w i l l hopefu l ly provide some evidence as to whether subspec ia t ion has occurred between the A rc t ic and Montana forms o f Thymallus OXtC t 1IoU S . MATERIALS AND METHODS Sampling o f Populations D isc re te popula tions o f e i t h e r the Canadian form o r the Montana form of Thymallus avctious were chosen f o r a n a ly s i s . P opu la t i o n s , which accord ing to Montana Fish and Game s tock ing r e co rd s , were known not to be a mixture o f both forms were sampled as r e p r e s e n t a t i v e o f the Montana form. The Canadian a r c t i c popula t ion was assumed to be n a t i v e , but des igna t ion o f any popula tion as a Montana form had to be confirmed from t r a n s p l a n t reco rds . The popula t ions used as r e p r e s e n t a t i v e s o f the Canadian form were taken from the Donnelly R iver , N.W.T. and Fuse Lake, Montana. The Donnelly River l i e s in the Mackenzie River dra inage and g ray l ing are na t ive to i t s waters (McPhail and Lindsey 1970). Fuse Lake in Grani te County, Montana, i s an a lp in e lake which had no n a t iv e f i s h fauna. Fuse Lake was s tocked in 1930 with g ray l ing from the Saskatche wan River d ra inage (S ta te Fish and Game Records) with no subsequent p l a n t ing s , thus i t i s a pure popula tion o f the Canadian form. The Saskatchewan River popula tion in tu rn was der ived from a Canadian A rc t ic g ray l ing popula t ion (Lindsey 1956). The Montana form was sampled from Grebe and Wolf Lakes popula t ions in Yellowstone National Park. Grebe and Wolf Lakes a re connected by f i v e hundred meters o f the Gibbon River which flows from Grebe Lake 20 in to Wolf Lake. Both lakes have i n l e t and o u t l e t spawning adapted popula t ions o f g ray l ing . In sampling these popu la t ion s , only the 1 o u t l e t spawning Wolf Lake popula tion and the i n l e t spawning Grebe Lake popula t ion were sampled in o rde r t h a t any gene t i c d i f f e r en c e s a s soc ia ted with t h i s spawning behavior could be e s t imated in the e l e c t r o p h o r e t i c survey. The g ray l ing were c o l l e c t e d e i t h e r by ang l ing o r by use o f an e l e c t r i c backpack shocker . The number o f i n d iv idua l s c o l l e c t e d , the d a te , the method, and the l o ca t ion s i t e a re l i s t e d in Table I . Upon cap tu re the weight and length o f the f i s h were recorded. Blood samples were taken by making a long i tud in a l i n c i s io n from the isthmus to the abdominal region o f the f i s h , opening the p e r i c a r d i a l sac with an i n c i s io n and removing approximate ly I ml o f blood. The blood was placed in a p l a s t i c tube and e i t h e r c en t r i fuged a t 5000 g f o r approximate ly 3 minutes when done in the f i e l d , or the tube of blood was placed on ice and t r a n spo r t e d to the l a bo ra to ry where c en t r i fug ing was done. A f te r c e n t r i f u g a t i o n th e serum was s epa ra ted from the blood c e l l s , s to red in a microfuge tube and both were immediately f rozen . Tissue samples o f the l i v e r , muscle, h e a r t and eye were taken and placed immediately on dry ice and were t r a n s f e r r e d to a f r e e z e r maintained a t -50°C upon r e tu rn to the l abo ra to ry . A f te r removal Table I . Col lec t ion data f o r the four popula tions of Thymallus arotiaus. Population Date Number o f Ind iv iduals Ancestral Stock Grebe Lakea Y.N.P. 6/25/75 30 Madison River System 6/11/76 30 6/17/76 18 6/7/77 22 Wolf Lakeb Y.N.P. 6/25/75 8 Madison River System 7/22/75 12 6/10/76 10 5/24/77 22 5/31/77 8 Donnelly River N.W.T. 9/1/76 44 Native Fuse Lake Mont. 8/15/75 19 Mackenzie River a I n l e t Creek ^Outle t Creek 22 of t i s s u e samples the sex o f the ind iv idua l was noted when i t could be de termined, otherwise i t was c l a s s i f i e d as immature. Sample P repara t ion Tissue e x t r a c t s were prepared by gr ind ing the t i s s u e in an equ iva ­ l e n t volume o f bu f f e r (.01 M I r i s ; .001 M EDTA; 5 x 10 ^ M NADP; pH ad jus ted to 6 . 8 ) , in a g la s s homogenizer. The homogenate was then cen t r i fuged a t 15,000 g f o r 20 minutes in a r e f r i g e r a t e d c en t r i f u g e maintained a t -10°C. The supe rna tan t was then removed fo r e l e c t r o ­ phores is o r s to red a t -50°C f o r l a t e r use. E lec t rophore s i s Horizontal s t a r ch gel e l e c t ro p ho r e s i s was used in th e e l e c t r o ­ p ho re t i c a n a ly s i s of p r o t e i n s . The s t a r ch ge ls were 11.2% hydrolyzed s t a r ch (E le c t ro s t a r c h C. Madison, Wisconsin). The s t a r ch was mixed thoroughly with the app rop r i a t e amount o f b u f f e r in a s ide arm f l a s k and placed on a hot p l a t e with a magnetic s t i r r e r , with add i t io n a l hand shaking , u n t i l the so lu t io n bo i led . The r a t e o f s t i r r i n g was inc reased as the v i s c o s i t y o f the f l u i d inc reased to keep the f l u i d homogeneous. A vacuum was then app l ied to the f l a s k f o r approximate ly 60 seconds to remove a i r bubbles. The hot s t a r c h so lu t io n was poured on g la ss p l a t e s 25 cm by 18 cm. P lex ig la s s s t r i p s 1.5 cm wide and I cm t h i c k , he ld in place by la rge paper clamps, were used to form 23 the edges o f the g e l . A f te r an i n i t i a l coo l ing (I to 2 hours a t room temperature) the gels were covered with p l a s t i c wrap w i thout t rapp ing any bubbles and s to red in a r e f r i g e r a t o r . Gels were used f o r e l e c t r o ­ phores is any time up to 24 hours l a t e r . P r io r to a pp l i c a t i o n o f samples to the g e l , 26 sample s l o t s . 5 x 1 cm were made across the gel by a s l o t former. This allowed each sample s l o t to be completely s epa ra ted by .5 cm o f ge l . A f i l t e r paper wick (approximate ly I cm x .5 cm) was soaked in a sample supe rna tan t , b l o t t e d on f i l t e r paper to remove excess , and placed in the sample s l o t by the use o f small fo rceps . P l a s t i c bu f f e r t r a y s (15 cm x 5 cm x 3 cm) were f i l l e d with 250 ml bu f f e r . Disposable c lo th s (Handy Wipes) 25 cm wide, were used as the e l e c t rod e sponges. The sponges were placed 3 cm from the ends o f the g e l , and the e n t i r e top o f the appara tus was covered with p l a s t i c wrap. A d i r e c t e l e c t r i c a l c u r r e n t , the vo l tage o f which va r ied as to bu f f e r system used was app l ied to the g e l . The gel was cooled by a pan of ice suspended 2 mm over the e n t i r e gel su r face by p l e x ig l a s s s t r i p s . E lec t ropho re s i s was continued u n t i l a marker (Bromophenol Blue) reached the anodal sponge. The bu f f e r systems employed, vo l tage used, and length o f run a re l i s t e d in the Appendix. 24 Af te r e l e c t ro pho r e s i s was completed, the ge ls were s l i c e d in to .20- .25 cm th ick s l i c e s by a s t a i n l e s s s t e e l b lade . The top s l i c e was always d iscarded due to d i s t o r t i o n and the bottom 3 s l i c e s were s t a in ed by the app rop r ia te procedure f o r v i s u a l i z a t i o n o f the p ro te in s de s i r ed . The s t a i n in g procedures f o r d i f f e r e n t p ro te in systems a re l i s t e d in the Appendix. A f te r the app rop r i a t e t ime f o r adequate development of the zymogram, the ge ls were washed th r e e t imes in d i s t i l l e d water and f ixed in a me thano l /w a te r / a c e t i c ac id (5 /5 /1 ) s o lu t ion . Permanent records o f the zymograms were made by keeping a w r i t t e n ■ record o f the r e s u l t s , and photographing each gel on 35 mm Kodak High Contras t Copy or Panotomic X f i lm . The l a t t e r type appeared to produce b e t t e r r e s u l t s . The f ixed ge ls were covered with Saran Wrap and r e f r i g e r a t e d fo r l a t e r comparison with the photographs. Q ua l i t a t iv e Analysis Isozymes a re defined as mu l t ip l e molecular forms o f an enzyme occurr ing in a s ing le ind iv idua l o r in d i f f e r e n t members o f the same spec ies (Markert 1968). Such isozymes may occur to g e th e r in the same c e l l , but th e re may a l so be marked d i f f e r en ce s in isozyme p a t t e r n s between t i s s u e s (Harr is 1975). Several au thors (ManweTl and Baker 1970, Harr is 1976) have d iscussed the f a c t t h a t isozyme systems may be generated in a v a r i e t y o f ways. The var ious causes o f isozymes may 25 be c l a s s i f i e d in to 3 main c a t e go r i e s (Harr is 1969, 1975; Hopkinson and Harr is 1971): I) occurrence o f mu l t ip l e gene loc i coding f o r s t r u c ­ t u r a l l y d i s t i n c t polypeptide chains o f the enzyme; 2) occurrence o f mu l t ip le a l l e l i sm a t a s i n g l e locus determin ing s t r u c t u r a l l y d i s t i n c t vers ions o f a p a r t i c u l a r po lypep t ide cha in ; 3) occurrence o f s o - c a l l e d "secondary isozyme formation due to pa s t t r a n s l a t i o n a l mod i f ica t ion s of the enzyme s t r u c t u r e . Furthermore, apparent v a r i a t i o n observed on zymograms o f s ta r ch gel e l e c t r o p ho r e s i s may be a r t i f a c t s due to s to rage and p re se rva t ion o f mate r ia l (Eppenberger e t at. 1971), or i t may be the r e s u l t o f d i f f e r e n t i a l a c t i v a t i o n o f genes due to env i ron ­ mental cond i t ions (Hochachka and Somero 1971). S t r ing en t c r i t e r i a must be used in ana lyz ing biochemical v a r i a ­ t ion o f zymograms before one can assume t h a t th e re i s a g ene t i c ba s i s f o r the v a r i a t i o n . The s t r o n g e s t data f o r de te rmin ing i f the v a r i a ­ t i o n has a gene t i c bas is comes from breeding exper iments and the subsequent a n a ly s i s o f progeny from paren ts having known biochemical d i f f e r e n c e s . In the absence o f such d a t a , as i s the case in the p re sen t s tudy , o th e r c r i t e r i a must be imposed as sugges ted by var ious au thors (U t te r e t a l . 1973, Avise and Smith 1974, .Gall e t a l . 1976). The c r i t e r i a used in the p re sen t s tudy to v e r i f y the gene t i c ba s i s o f observed v a r i a t i o n were: 26 1) The banding p a t t e r n observed was t y p ic a l f o r the presumed s t r u c t u r e o f the enzyme found in c lo se ly r e l a t e d sp ec ie s . 2) The p a t t e rn was i n t e r p r e t a b l e on the b a s i s of a simple gene t ic hypo thes i s ; with t y p ic a l homozygotes and he te rozygotes being expressed. 3) The Observed genotypic ,class p ropo r t ions were in c lo se ag ree ­ ment with those expected on the bas is o f Hardy-Weinberg equ i l ib r ium ; any s i g n i f i c a n t d ev ia t ion s from the p red ic t ed values must be exp la ined . 4) P a t te rn s must be r epea tab le upon subsequent sampling o f the same i n d i v i d u a l , with p a r a l l e l express ion in o th e r t i s s u e s to confirm any polymorphism. Var ia t ion which d id no t conform to a l l o f th e se c r i t e r i a could not be conc lus ive ly v e r i f i e d to be g ene t i c . However, i f the evidence presented was s t rong ly sugges t ive o f a gene t i c b a s i s , i t was included in the a n a ly s i s . Since isozymes may have d i f f e r e n t i a l t i s s u e exp re s s ion , severa l d i f f e r e n t t i s s u e s were analyzed to determine the p a t t e r n o f such express ion . The t i s s u e s examined f o r a given enzyme system are summarized in Table 2. This allowed the de te rmina t ion o f an added number o f loc i whose p ro t e in may be expressed in one t i s s u e and not another (eg. LDH-5 locus ) . A v a r i e t y o f bu f f e r systems were employed in e a r l y sc reen ing runs s ince v a r i a t i o n may be expressed in one b u f f e r Table 2. Pro te ins surveyed, t i s s u e s examined, and bu f f e r systems employed in e l e c t r o ­ phore t ic ana ly s is o f Thymallus a ra ticu s . Abbre- Tissue Buffer Prote in E.C. No. v ia t ion Liver Muscle Heart Eye Serum System Alcohol Dehydrogenase (1 .1 .1 .1 ) (ADH) + C Alpha-glycerophosphate Dehydrogenase ( I . I . I . 8) (AGPD) C,D,F Este rase ( 3 . I . I . I ) (EST) + I Glucose-6-Phosphate Dehydrogenase (1 .1 .1 .49 ) (G-6-PD) + + + + A,F Glutamate Oxaloacetate Transaminase ( 2 . 6 . I . I ) (GOT) + + + + C1H Hexose-6-Phosphate Dehydrogenase (1 .1 .1 .47 ) (H-6-PD) + + + + A,F Hexokinase ( 2 . 7 . I . I ) (HK) + G I s o c i t r a t e Dehydrogenase (1 .1 .1 .42 ) (IDH) + + B1D Lactate Dehydrogenase (1 .1 .1 .27 ) (LDH) + + + + + F1G1H Malate Dehydrogenase (1 .1 .1 .37 ) (MDH) + + + + + A,B,D Malic Enzyme (1 .1 .1 .40 ) (ME) + + A,B Table 2. (Continued) Protein E.C. No. Abbre­ v ia t ion Tissue Liver Muscle Heart Eye Serum Buffer System Phosphoglucomutase ( 2 . 7 . 5 . I) (PGM) + + A,E Trans fe r r in (TFN) + I Tetrazolium Oxidase (TO) + + A1F Serum Prote ins (SP) + I Sorbi to l Dehydrogenase (1 .1 .1 .14 ) (SDH) + H Xanthine Dehydrogenase ( I . 2 .3 .2 ) (XDH) + + A1F 29 but not ano the r (U t te r e t a l . 1973). The d i f f e r e n t b u f f e r systems employed f o r r e s o lu t ion o f a given p ro te in a re l i s t e d in Table 2. Salmonid f i s h a re be l ieved to have undergone ex ten s ive gene dup l ica t ion and, t h e r e f o r e , may be t e t r a p l o i d organisms (Ohno 1969). Grayling a re salmonids , hence in the gene t i c i n t e r p r e t a t i o n o f e l e c t r o p h o r e t i c p a t t e r n s , i t i s impor tant to r e a l i z e t h a t th e r e may be two or more gene loc i which determine the primary s t r u c t u r e of p ro t e in s . The presence o f such dup l ica ted loc i has been found to e x i s t in many salmonids (A l lendorf e t a l . 1975, Bai ley e t a l . 1970, Morrison and Wright 1966, Wolf e t a l . 1970). The re fo re , g ray l ing would be assumed to have s im i l a r l y dup l ica ted l o c i , complicating the a n a ly s i s o f zymogram p a t t e r n s . Genetic i n t e r p r e t a t i o n was f u r t h e r c l a s s i f i e d by determining i f the loc i were dup l i c a ted which exp la ined observed branding p a t t e rn s o f these l o c i . This allowed v a r i a t i o n to be a t t r i b u t e d to e i t h e r mu l t ip l e loc i o r to mu l t ip l e a l l e l e s a t a s ing le locus . All o f the above p o s s i b i l i t i e s were considered and the c r i t e r i a imposed before the gene t ic b a s i s o f v a r i a b i l i t y was assumed. Nomenclature The system o f nomenclature fo llows t h a t o f Richmond (1972) and Prakash, Lewontin, and Hubby (1969). Each locus was named using an abb rev ia t ion o f the p ro te in name. When p ro te in s o f two o r more loc i 30 with i d e n t i c a l s u b s t r a t e s p e c i f i c i t i e s were observed, the loc i were numbered accord ing to the migra t ion r a t e o f the p ro te in products from the o r i g i n , i . e . , the loc i whose p ro te in had the s lowes t migra tion was ass igned numeral one, the next f a s t e s t the numeral two, and so on. When a l l e l i c v a r i a t i o n was found a t a lo cus , the most common a l l e l e was ass igned a number 1.00 and a l l o th e r a l l e l e s were ass igned numbers t h a t r ep resen ted t h e i r p ro t e in s mig ra t ion d i s t a nce r e l a t i v e to the most common a l l e l e ' s p r o t e i n , i . e . , i f a p ro t e in migrated a d i s t a n ce h a l f as f a r , i t s a l l e l e would be labe led 0.50 o r i f one migrated a d i s tance one and a h a l f t imes as f a r , i t s a l l e l e would be des igna ted 1.50. RESULTS E lec t ropho re t ic Phenotypes o f Monomprphic P ro te in s Lac ta te dehydrogenase - (LDH) i s a te t r ame r composed o f subun i ts o f equal s i z e (Appel!a and Markert 1961). LDH isozymes have been resolved e l e c t r o p h o r e t i c a l Iy i n to two main types des igna ted A and B (Markert 1962). In mammals thus f a r s t u d i e d , f i v e p r in c ip a l isozymes a re u sua l ly found which r e s u l t from the random combination o f these subun i ts A and B, i n to a l l po s s ib le t e t r am e r i c s t r u c t u r e s . Shaw and Barto (1963) provided g ene t i c conf i rmat ion o f the hypothesis t h a t the subun i ts were under d i s t i n c t gene t i c con t ro l by showing t h a t each was c on t ro l l e d by s epa ra te gene l o c i . An a dd i t io n a l isozyme, des igna ted C, has been observed in mature t e s t i s e x t r a c t s o f mammals and b i rd s (Blanco and Zinkham 1963, Goldberg 1963). I t has subsequently been shown to be coded by a sepa ra te locus (Blanco e t a t . 1964). Isozyme p a t t e r n s o f t e l e o s t f i s h provide evidence o f an add i t iona l LDH l o c u s , former ly d e s ig ­ na ted E, in these animals (Markert and Faulhaber 1965, Whitt 1969, 1970, Markert and Holmes 1969, ShakTee e t a t . 1973). This locus i s now considered t o be homologous to the C locus of b i rd s and mammals, with t i s s u e express ion varying with the f i s h s tud ied (Markert e t a t . 1975). Genetic s tu d i e s o f e l e c t r o p h o r e t i c v a r i a n t s have e s t a b l i s h e d the ex i s t ence o f t h i s locus (Whitt e t a t . 1971). VThe isozyme i s expressed predominantly in nervous t i s s u e , e s p e c i a l l y in the r e t i n a o f the eye (Goldberg 1966, Whitt 1969, 1970, Whitt and Horowitz 1970, 1972). In the case o f Sa1Imonid f i s h , e s p e c i a l l y the t r o u t , a complex p a t t e r n o f more than f i f t e e n isozymes i s obse rvab le , with g ene t i c s tud ie s showing t h a t t r o u t LDH i s determined by a t l e a s t f i v e d i s ­ t i n c t loc i (Morrison and Wright 1966, Morrison 1970, Massaro and Markert 1968, U t t e r e t a t . 1973). Re la t ive to t h i s obse rva t ion i s the hypothesis o f Ohno e t at . (1968) , based on cy to log ic a l ev idence , t h a t salmonids a re t e t r a p l o i d s . This i n d i c a t e s t h a t th e re has been dup l ica t ion and subsequent divergence o f A and B l o c i . The v a l i d i t y o f the ex i s t ence o f dup l ica ted loc i i s supported by the molecular hyb r id i za t ion and immunochemical, s t u d i e s o f Massaro and Markert (1968). The re fo re , the loc i in salmonids a re comprised o f the dup l ica ted A (A and A1) and B (B and B1) loc i and an add i t iona l C locus . The complex zymogram p a t t e r n s o f LDH isozymes a re exp la ined by: f iv e isozymes con ta in ing A and A1 s ubun i t s , f i v e isozymes con­ t a i n in g B and B1 subuni ts and hybrid isozymes con ta in ing B, B1 and C subun i ts . 1 The var ious loc i have d i f f e r e n t i a l t i s s u e exp re ss ion . The B and B1 a re expressed in most t i s s u e s (see Massaro and Markert 1968), except the l i v e r where the B1 predominates (U t t e r and Hodgins 1972). 32 33 The A and A' loc i a re expressed p r im ar i ly .in the s k e l e t a l muscle (Massaro and Markert 1968, U t t e r e t a l . 1973). The C locus i s expressed in th e eye and o th e r neural t i s s u e (Horowitz and Whitt 1972). The isozyme p a t t e r n s found in T. avctious were ty p ic a l o f t h a t found in o th e r salmonids (Fig. 3) . Five loc i des igna ted LDH-I, LDH-2, LDH-3, LDH-4, and LDH-5 (equ iva len t to A, A ' , B, B1 and C, r e sp ec t iv e ly ) in o rde r o f in c re a s ing anodal m ig ra t ion , were expressed In muscle t i s s u e , 5 isozymes r e s u l t i n g from the combination o f LDH-I and LDH-2 subun i ts and 5 isozymes r e s u l t i n g from the combination o f LDH-3 and LDH-4 subuni ts were expressed .on LDH zymograms. The A group predominated as expected f o r salmonids. In h e a r t , serum and eye, f iv e isozymes r e s u l t i n g from the combination o f LDH-3 and LDH-4 subuni ts (B group) were expressed . An add i t io n a l locus LDH-5 was expressed in eye t i s s u e which i s the C locus common to salmonids (Horowitz and Whitt 1972). In l i v e r , the LDH-3 locus predominated, e xh ib i t i n g a s in g l e band in most b u f f e r systems. However, in bu f f e r system A f iv e isozymes could be re so lv ed , thus both the LDH-3 and LDH-4 loc i a re expressed in the l i v e r . The predominance o f the LDH-3 locus (B) i s in c o n t r a s t t o the predominance o f the LDH-4 (B ' ) locus in the l i v e r o f o th e r salmonids (U t te r and Hodgins 1972, U t t e r e t a t . 1973). 34 L E M M L b. + — LDH-4 ^LDH-3 c. Figure 3. Lactate Dehydrogenase (LDH). Tissue d is tr ib u tio n and the e f fe c ts o f d if f e re n t bu ffe rs on re so lu tio n o f the LDH isozymes. a. L iver, eye and muscle t is s u e o f the same f is h . Buffer system H. b. Muscle and l iv e r t is s u e from the same f is h . Buffer system G. c. Liver t is s u e from th ree in d iv idu a ls . Buffer system A. 35 No a l l e l i c v a r i a t i o n was found a t any o f the loc i in any o f the popula t ions examined. Ind iv idua ls were homozygous a t a l l l o c i , t h e r e f o r e , eleven isozyme types were expressed . The fewer number o f isozymes r e s u l t e d from the apparent lack o f h e te ro te t ramer s being formed between the LDH-5 and LDH-3 o r LDH-4 l o c i . The number o f LDH isozymes in T. ape ticu s i s evidence t h a t gene dup l i c a t ion has taken place a t the A and B loc i o f LDH. Malate dehydrogenase (MDH) - Two major c l a s s e s o f malate dehy­ drogenase isozymes a re found to e x i s t in v e r t e b r a t e s , the mitochondria l form and the superna tan t (o r cytoplasmic) form (K i t to and Kaplan 1966, Siegal and England 1961). Both have a dimeric s t r u c t u r e and a re coded by d i s t i n c t nuc lea r genes (Shows e t a l . 1970, Wheat e t at. 1971). Three types o f supe rna tan t MDH isozymes AA, AB, BB, coded by 2 gene loc i A and B, are found in many spec ie s o f f i s h (Wheat and Whitt 1971, Clayton e t al . 1971). The i n v e s t i g a t i o n s o f Bai ley e t a t . (1970) i nd ic a ted t h a t t h i s was the case in salmonids. In the s a l ­ mon i d s , not only i s th e re evidence o f gene t i c v a r i a t i o n of. the B form, o f superna tan t MDH (Sy ln1ko 1972, U t t e r e t a l . 1973, Clayton e t al. 1975, Bai ley e t o l . 1970, AspinwalI 1974), but th e re i s a l s o evidence o f dup l ic a t ion o f the B locus (Bailey e t a l . 1970, U t t e r and Hodgins 1972, A l lendo r f 1973). Bai ley e t a l . (1970) provided evidence from 36 isozyme dosage s tud ie s which sugges ted t h a t the A locus i s a l s o dup l ica ted in the brown t r o u t {Salmo t r u t t a ) , while Aspinwall (1974) suggested t h a t t h i s was a l so t ru e f o r pink salmon (Oncorhynahus, gorbusoha). However, t h i s has not been found in o th e r sa l mom" d spec ies (S y ln 1ko 1976, Clayton e t a l . 1975, A l lendo r f 1973). There i s t i s s u e s p e c i f i c express ion o f the A and B loci with the B form predominating in s k e l e t a l muscle and the A form predom­ ina t ing in the l i v e r (Clayton e t a l . 1975, A l lendo r f e t a l . 1973, Al lendorf 1973, U t te r and Hodgins 1972, Bai ley e t a l . 1970). Massaro (1973) s tud ied the MDH isozymes o f g ray l ing and repo r ted t h a t they possess th r e e major isozymes o f supe rna tan t MDH in s k e l e t a l muscle and eye t i s s u e , which may correspond to the AA, AB, and BB isozymes p re s en t in o th e r salmonids as r epo r ted by Bai ley e t a l . (1970). In the p re sen t s tudy , t i s s u e samples o f l i v e r , s k e l e t a l muscle, h e a r t muscle, eye and serum were surveyed for. supe rn a tan t MDH a c t i v i t y . The r e s u l t s a re shown in Figure 4. A s in g l e band o f s t rong i n t e n s i t y was found in l i v e r samples, whereas 3 bands were expressed in a l l o t h e r t i s s u e s surveyed. I t i s p o s tu l a t ed t h a t th e re a re two loc i MDHg-I and MDH5 -E , corresponding to the A and B l o c i , r e spec ­ t i v e l y , o f o t h e r salmonids, coding fo r supe rna tan t MDH isozymes in T. arotiaus. The A form, encoded by the MDH5-I l o c u s , i s predominant in the l i v e r , while both forms A and B, (B encoded by the MDH5-E 37 Figure 4. Tissue d is tr ib u tio n of Malate Dehydrogenase (MDH) from the same f ish . Samples are o f l iv e r , muscle, h e a r t , serum and eye t is s u e . The MDH isozyme near the o r ig in is common to a l l t is su e s but s ta in s weakly in muscle, h ea rt and serum samples. MDHs -I predominates in l iv e r t is s u e . Both MDH5-I and MDHg-2 are more equally expressed in muscle, h ea rt and serum tis s u e . 38 locus) a re expressed in the o th e r t i s s u e s surveyed. The th r e e banded phenotype seen f o r t h i s d imeric enzyme i s b e s t exp la ined by the ran - . dom combination o f subun i t products from two loc i with d i f f e r e n t a l l e l e s . This r e s u l t s in a f ixed he terozygote e f f e c t with every ind iv idua l e x h ib i t i n g the th r e e banded phenotype. No v a r i a t i o n a t e i t h e r locus was found in any o f the popula t ions s tud ied . Since g ray l ing possess no polymorphism a t the loc i coding fo r both the A type and B type s u b u n i t s , i t was not po s s ib le to determine i f d up l i c a t ion has taken place a t t h e se two l o c i . A fou r th band o f a c t i v i t y was a l so found on zymograms s t a i n ed fo r MDH a c t i v i t y . This isozyme had a s h o r t e r mig ra t ion d i s t a nce and weaker s t a i n in g i n t e n s i t y than the o th e r forms. This isozyme i s be l ieved to be the mitochondria l form o f MDH (des igna ted MDHm) based on c o r r e l a t i o n with zymograms o f o th e r salmonids (Sy ln 'ko 1976, Aspinwall 1973, Bai ley e t a t . 1970, Bai ley e t a t . 1969), and i s con­ t r o l l e d by a s epa ra te gene locus (d iscussed p rev iou s ly ) . No v a r i a t i o n was observed f o r t h i s isozyme in any o f the popu la t ions s tud ied . Glu tamate-oxa loace ta te t ransaminase (GOT) - There a re two d i s ­ t i n c t forms o f g lu tama te -oxa loace ta te t ransaminase in v e r t e b r a t e c e l l s (Moore and Lee 1960), both o f which have a d imeric s t r u c t u r e (DeLorenzo and Ruddle 1970). One o f the forms o f GOT i s found in the mitochon­ d r i a l f r a c t i o n while the o th e r one occurs in the supe rna tan t (or 39 cytoplasmic) f r a c t i o n o f the c e l l . The supe rna tan t form migra te s anodally a t a neu t ra l pH, whi le the mitochondria l form migra te s ca thoda l Iy (Schmidtke and Engel 1972). The supe rna tan t form o f GOT has been shown to be coded f o r by two loc i in seve ra l f i s h spec ies (Schmidtke and Engel 1972). In sa lmonids , GOT has been repor ted to be coded by two disomic loc i in. brown t r o u t (Schmidtke and Engel 1972), chum salmon (A l lendorf e t a l . 1975, May e t a t . 1975) and c u t t h r o a t t r o u t (A l lendorf and U t te r 1976). In the p re s en t s tudy , g ray l ing were surveyed fo r GOT a c t i v i t y in l i v e r , s k e l e t a l muscle, h e a r t muscle, and eye t i s s u e : The r e s u l t s a re shown in Figure 5. The b u f f e r system employed in the s epa ra t ion o f GOT isozymes had a high pH ( 8 . 6 ) , which r e s u l t s in the anodal migra tion o f both forms (mitochondrial and s u p e r n a t a n t ) . The r e s u l t s i n d i c a t e t h a t th e re a re a t l e a s t two loc i (GOT5-I and GOT5 -2) coding f o r the supe rna tan t form o f GOT and two loc i (GOTm-I and GOTm- 2) coding f o r the mitochondria l form. There a re two bands o f a c t i v i t y f o r the mitochondria l form with no heterodimer formed between the two. This r e s u l t i s b e s t expla ined by the presence o f 2 loc i (GOTm-I and G0Tm~2) coding f o r d i s t i n c t polypeptide subun i ts which do not i n t e r a c t with one ano ther to form a heterodimer c on s i s t i n g o f a subun i t from each locus . No v a r i a t i o n was observed a t e i t h e r locus in any o f the popula t ions surveyed. 40 Figure 5. Glutamate oxaloaceta te transam inase (GOT) t is s u e d i s t r i ­ bu tion . a. Liver samples from th ree f is h with expression o f two GOTs and two GOTm lo c i . b. Tissue d is tr ib u tio n of GOT isozymes from one f ish ; eye, muscle, l iv e r . GOTm isozymes d id not s ta in on th is ge l. 41 The supe rna tan t GOT exh ib i t e d d i f f e r e n t p a t t e r n s in the var ious t i s s u e s examined. In the l i v e r a th r e e banded phenotype with asymmetrical s t a i n i n g i n t e n s i t y was found in a l l i n d iv id u a l s . This p a t t e rn may be exp la ined by the presence o f two loc i with d i f f e r e n t a l l e l e s coding f o r subuni ts o f d i f f e r e n t e l e c t r o p h o r e t i c mob i l i ty . This f ixed he terozygote e f f e c t i s evidence fo r the presence o f a dup l ica ted locus (A l lendorf e t d l . 1975). In eye t i s s u e a s im i l a r th re e banded p a t t e r n was the phenotype o f a l l i n d i v i d u a l s , however, the asymmetry o f s t a i n in g i n t e n s i t y was in the oppos i te d i r e c t i o n (Figure 5b). This p a t t e rn again sugges ts a dup l ic a ted locus . The asymmetry of i n t e n s i t y cannot be expla ined by the p o s tu l a t e t h a t one o f the loc i i s monomorphic f o r the common a l l e l e while the o th e r i s polymorphic s in ce : I ) a l l i n d iv id u a l s express the same p a t t e r n which would not be expected i f the locus was polymorphic (some would be symmetr ical) , and 2) th e re i s oppos i te asymmetry in a d i f f e r e n t t i s s u e of the same ind iv id u a l . The lack o f p a r a l l e l express ion between eye and l i v e r sugges ts t h a t the p a t t e r n i s the r e s u l t o f d i f f e r e n t i a l a c t i v a t i o n o f the two loc i in these d i f f e r e n t t i s s u e s . In muscle, only one band o f supe rna tan t GOT a c t i v i t y was expressed. The band had the same e l e c t r o p h o r e t i c mob i l i ty as the GOTg-I homozygote in l i v e r . There i s evidence in some salmonids t h a t t h i s band may rep re sen t a d i s t i n c t locus (A l lendorf personal 42 comm., A l lendo r f and U t te r 1976). However, the lack o f v a r i a t i o n a t t h i s locus does not al low the conf i rmat ion o f t h i s p o s tu l a t e in g ray l ing . The re fo re , the conse rva t ive e s t ima te o f two supe rna tan t GOT loc i and two mitochondria l GOT loc i was used in the p re s en t study. Alcohol dehydrogenase - (ADH) has been shown to have a dimeric s t r u c tu r e in v e r t e b r a t e s (Bu t l e r e t d l . 1969). I t i s b e l ieved to be under the con t ro l o f a s i n g l e gene locus in salmonids (A l lendorf e t dl . 1975). The enzyme in t r o u t appears to be n ega t iv e ly charged, migrating to the cathode. In most salmonids s t u d i e d , ADH appears as a s in g l e band with l i t t l e o r no v a r i a t i o n w i th in the spec ie s (Kr is t iansson and McIntyre 1965, Gall e t d l . 1977, U t t e r e t d l . 1973, Al lendorf e t d l . 1975). In T. areticus l i v e r samples were examined f o r ADH a c t i v i t y . In a l l i n d iv idua l s ADH was expressed as an i d e n t i c a l s i n g l e band o f a c t i v i t y , m ig ra t ing ca thoda l ly . On the b a s i s o f th e se r e s u l t s i t was po s tu la t ed t h a t ADH i s encoded by a s in g l e locus in g ray l ing . The ADH locus was monomorphic and i d e n t i c a l in a l l popula t ions surveyed with no v a r i a t i o n between popu la t ions . Xanthine dehydrogenase - (XDH) i s r ep re sen ted in salmonids by a s ing le band o f a c t i v i t y which migra tes anodal ly (K r i s t i an s son e t at . 1976, A l lendo r f e t d l . 1975, A l lendo r f 1973). Since no v a r i a t i o n 43 e x i s t s a t t h i s locus in the spec ie s s tu d i e d , no conclus ions can be made as to whether XDH i s encoded by a s in g l e locus o r dup l ic a ted locus (A l lendorf e t a t . 1975). In T. arct-ious a s in g l e band o f XDH a c t i v i t y was found which migrated anoda l ly . All popu la t ion s were monomorphic with no v a r i a ­ t i o n de tec ted between popu la t ions . I t i s assumed t h a t the enzyme is coded f o r by a s ing le locus . However, the p o s s i b i l i t y of two loc i with i d e n t i c a l e l e c t r o p h o r e t i c gene products cannot be excluded. Sorb i to l dehydrogenase - (SDH) i s presumed to have a t e t r am e r i c s t r u c t u r e in v e r t e b r a t e s (OptlHof 1969, Opt1Hof e t a l . 1969, Engel e t al . 1970). Engel e t a l . (1970) proposed t h a t . t h i s i s a l s o the s t r u c tu r e o f SDH found in rainbow t r o u t , and has r epo r ted the presence o f a polymorphism. In v a r i a n t mult i-banded phenotypes have been repor ted in var ious o th e r salmonid spec ie s (May e t a l . 1975, Khanna e t a l . 1975, A l lendorf e t a l . 1975, U t t e r e t a l . 1973). The p a t t e rn s in these spec ies i s i n d i c a t i v e o f two disomic loc i f ixed f o r a l l e l e s coding f o r subun i ts o f d i f f e r e n t e l e c t r o p h o r e t i c mobi l i ty (Allendorf e t a l . 1975, U t t e r e t a l . 1973). This p o s tu l a t e i s in c o n t r a s t to t h a t o f Engel e t a l . (1970) which proposed a t e t r a som ic mode of in h e r i t a n c e fo r a s in g l e locus based on the phenotypic d i s ­ t r i b u t i o n o f isozyme p a t t e rn s with no breeding experiments performed. A l lendorf e t a l . (1975) on the ba s i s o f p re l im ina ry i n h e r i t a n c e 44 data o f a v a r i a n t in c u t t h r o a t t r o u t , provide f u r t h e r evidence in support o f the former p o s tu l a t e . In the p re s en t s tudy , g ray l ing exh ib i t ed a d i s t i n c t s i n g l e band phenotype o f SDH a c t i v i t y , which migrated anodal ly . No in t ropopu- l a t i o n a l o r in t e rpopu la t io n a l v a r i a t i o n was observed. This r e s u l t allows no conclus ions to be drawn as to the presence o f a dup l ic a ted locus in T. ope ticu s as proposed f o r rainbow t r o u t (A l lendorf e t a l . 1975). For the purposes o f t h i s s tudy the conse rva t ive e s t im a te o f one locus coding f o r SDH was used. I s o c i t r a t e dehydrogenase (mi tochondrial form) - (IDH) e x i s t s in d i s t i n c t mitochondria l (here des igna ted IDHm) and supe rna tan t (IDHg) forms which are determined by d i s t i n c t gene loc i in v e r t e b r a t e s (Henderson 1968). Wolf e t a l . (1970) , showed t h a t IDHffl manifes ts a dimeric s t r u c t u r e in e l e c t r o p h o r e t i c s tud ie s o f rainbow t r o u t {salmo gairdneri) . Engel e t al . (1971) showed t h a t in some d ip lo id groups of f i s h , a s in g l e gene locus presumably coded f o r IDHffl, whereas in some t e t r a p l o i d groups two d i f f e r e n t gene loc i were r e spon s ib le f o r the f ixed he terozygote p a t t e r n observed on zymograms. A l lendorf e t a l . (1975) repor ted t h a t in rainbow t r o u t {Salmo galvdnevi) IDHffl i s a l so rep re sen ted by th re e nonvar ian t bands i n d i c a t i n g the presence o f two monomorphic disomic loc i with common a l l e l e s coding fo r sub­ un i t s o f d i f f e r e n t e l e c t r o p h o r e t i c m o b i l i t i e s . IDHffl a c t i v i t y i s b e s t v i su a l i z ed on zymograms o f h e a r t muscle e x t r a c t s . 45 In g r a y l in g , h e a r t muscle e x t r a c t s were surveyed f o r IDHfil a c t i v i t y . IDHjfi phenotypes were rep resen ted by th r e e i n v a r i a n t bands which i n d i c a t e the presence o f two monomorphic disomic l o c i , IDHjfi-I and IDHjfi-Z, with common a l l e l e s coding f o r subuni ts o f d i f f e r e n t e l e c t r o p h o r e t i c m ob i l i t i e s . The p a t t e rn observed i s evidence t h a t the IDHjfi locus in T. arotious i s dup l ic a ted as presumed t o . b e the case in rainbow t r o u t (A l lendorf e t a t . 1975). All ind iv idua l s expressed the same phenotype in a l l popula t ions s tud ied . Alpha-glycerophosphate dehydrogenase - (AGPDH) i s a d imeric molecule f o r which gene t i c v a r i a t i o n has been desc r ibed in various spec ies o f f i s h (Aspinwall 1972, U t t e r and Hodgins 1972, AlTendorf e t a l . 1975, Engel e t a t . 1971, Johnson e t a l . 1970, McCabe e t at . 1970). Engel e t a l . (1971) proposed the ex i s t ence o f th r e e d i f f e r e n t gene loc i (A, B, and C) coding f o r AGPDH in the brown t r o u t {Salmo t ru tta) and the rainbow t r o u t {,Salmo gaivdneri) with the random combination o f subuni ts from a l l e l e s a t the var ious loc i accounting fo r the complex zymogram p a t t e r n s . Other au thors (AspinwalT 1972, U t t e r and Hodgins 1972) .have proposed the ex i s t en ce o f a s in g l e locus , two codominant a l l e l e system in o th e r salmonids on the bas is o f e l e c t r o p h o r e t i c r e s u l t s . Aspinwall (1972) sugges ted the presence o f a s ing le locus in salmonids may be due to a " s i l en c ing " o f dup l ica ted loc i in salmonids. However, A l lendo r f e t a l . (1975) 46 provided evidence t h a t the b u f f e r system employed in the e l e c t r o ­ phores is o f AGPDH determines the number o f isozymes and hence, the number o f loc i which a re d e tec ted . A low pH phosphate b u f f e r al lows the d e tec t ion o f an added number o f loc i which a re no t de tec ted with the use of a high pH t r i s - b o r a t e system. In the p re sen t s tudy , th r e e loc i (AGPDH-I, AGPDH-2 and AGPDH-3) with a l l e l e s coding f o r p r o t e in s o f d i f f e r e n t e l e c t r o p h o r e t i c m ob i l i t i e s a re po s tu la ted to e x i s t in T. arot-ious. The zymograms obta ined r e f l e c t the presence o f these th r e e loc i with a c t i v e dimers formed from the subuni ts o f the d i f f e r e n t l o c i , r e s u l t i n g in s ix isozymes being formed. Liver and muscle t i s s u e e x t r a c t s e xh ib i t e d p a r a l l e l express ion o f the i d e n t i c a l number o f isozymes. Examination o f t h i s isozyme system with the high pH bu f f e r system r e s u l t e d in the de tec t ion o f only the AGPDH-I locus . Fu r the r s tu d i e s using the low pH b u f f e r system r e s u l t e d in the d e te c t ion o f the two add i t iona l l o c i . These f ind ings a re s im i l a r to those o f Engel e t at. (1971) and r e f l e c t the d i f f e r en c e s which a re observed depending on the bu f f e r system employed as suggested by A l lendo r f e t a t . (1975). No a l l e l i c v a r i a t i o n a t any o f the AGPDH loc i was de tec ted in any of the popula t ions surveyed. Es te rase - The banding p a t t e r n s observed in l i v e r samples o f Thymallus arotiaus were in c o n s i s t e n t in number and i n t e n s i t y . Due 47 to the incons i s tency , the l i v e r e s t e r a s e s were not d e a l t with in any d e t a i l . The e s t e r a s e o f the serum was rep re sen ted by a s i n g l e i n v a r i a n t band in a l l popu la t ions . I t was assumed the band was r e p r e s e n t a t i v e of a s ing le gene locus. Hexokinase - (HK) has been repo r ted to be rep re sen ted by a s ing le band in rainbow t r o u t (A l lendorf 1973). L iver samples of T. arcticus surveyed fo r hexokinase a c t i v e l y exh ib i t e d an i d e n t i c a l s ing le band in a l l popula t ions examined. I t i s assumed the band i s r e p r e s e n ta t i v e o f a s ing le gene locus . E lec t ropho re t i c Phenotypes o f Polymorphic P ro te in s Tetrazolium oxidase ( Indophenol oxidase) - TO i s the de s igna t ion fo r an enzyme f i r s t desc r ibed by Brewer (1967) which ox id ized reduced t e t r a z o l i urn dyes r e s u l t i n g in an achromatic region a g a in s t the co lored background o f reduced dye on e l e c t r o p h o r e t i c zymograms. I n t r a s p e c i f i c v a r i a n t s o f TO were found to e x i s t in f i f t e e n spec ie s o f P a c i f i c ro ck f i sh [Sebastodes) (Johnson e t a t . 1970b). I n t e r ­ spec ies v a r i a t i o n o f TO has been repo r ted t o e x i s t in the salmonids (U t te r 1971, U t t e r e t at . T973, May e t a t . 1975). The ex i s t en ce o f i n t r a s p ec i e s TO polymorphisms have a l so been found in the rainbow t r o u t (U t te r 1971, U t t e r e t a t . 1973, Cederbaum and Yoshida 1972, U t t e r and Hodgins 1972, A l lendo r f 1973) and Chinook salmon (U t te r 48 1971, K r i s t i an sson and McIntrye 1976). The p a t t e r n s observed appear to r e f l e c t one locus with two a l l e l e s encoding a d imeric p ro t e in (U t te r 1971, Cederbaum and Yoshida 1972). In T. aretieus ind iv idua l s e xh ib i t e d e i t h e r one zone o r th r e e zones of a c t i v i t y (Figure 6) s im i l a r to the p a t t e r n s found in o th e r salmonids (d iscussed p rev iou s ly ) . P a r a l l e l t i s s u e express ion was found in l i v e r and muscle e x t r a c t s o f the same in d iv id u a l . No in te rpopu la t io n a l v a r i a t i o n was found. The common a l l e l e (TO^" ^ ) , and a v a r i a n t a l l e l e ( t O " ^ ) , were found in th e Donnelly, Wolf and Grebe popu la t ion s . Homozygotes (1 .00 /1 .00) f o r the common a l l e l e expressed a s i n g l e band o f a c t i v i t y which migrated the f a r t h e s t anodal ly (Fig. 6 ) , he te rozygo tes (1 .00 / .50) e xh ib i t e d a 3 banded p a t t e r n with a h e t e r o d imeric zone of g r e a t e r i n t e n s i t y , and homozygotes ( . 5 0 / . 5 0 ) f o r the v a r i a n t a l l e l e exh ib i ted a s in g l e band with t h e . s l ow e s t m ig ra t ion . I t i s assumed t h a t the p a t t e r n s r e f l e c t a s in g l e lo cu s , two codominant a l l e l e system s im i l a r to t h a t suggested f o r rainbow t r o u t (U t t e r 1971, Cederbaum and Yoshida 1972). No v a r i a t i o n was found in the Fuse Lake popula t ion with a l l i nd iv idua ls being homozygous f o r the common a l l e l e . The lack of v a r i a t i o n in t h i s popula tion i s presumably a t t r i b u t a b l e to a founder e f f e c t . 49 1 2 3 4 5 Figure 6. Tetrazolium Oxidase (TO) polymorphism. Column I (0 .50/0 .50) homozygote. Column 2, 3, 4, (1 .00/0 .50) he te rozygo tes . Column 5 (1 .00/1 .00) homozygote. 50 Phosphoglucomutase - (PGM) isozyme v a r i a t i o n was f i r s t s tu d ied in human popula t ions by Spencer e t a t . (1964). Fu r the r gene t i c s tud ie s in humans showed the PGM a c t i v i t y could be d iv ided in to th ree zones , with each zone apparen t ly c o n t ro l l e d by an independent locus (Far r ing ton e t a t . 1968). Roberts e t a t . (1969) repo r ted f ind ing th r e e d i s t i n c t zones o f a c t i v i t y on PGM zymograms o f Satmo Qaivdnerii and po s tu la t ed the ex i s t ence o f th r e e l o c i , PGM-I, PGM-2 and PGM-3 ( in o rder o f in c re a s ing anodal migration o f the coded p ro t e in ) . Polymorphism o f PGM have been found in rainbow t r o u t (Roberts e t a t . 1969, U t t e r e t a t . 1972, 1973) and in var ious spec ie s of salmon (K r is t iansson and McIntyre 1976, May e t a t . 1975, U t te r e t a t . 1973, U t t e r and Hodgins 1970). In the p re sen t s tudy , l i v e r and muscle t i s s u e e x t r a c t s were surveyed f o r PGM a c t i v i t y . Three zones o f a c t i v i t y were de tec ted on PGM zymograms of l i v e r samples, a l l o f which migrate anodal Iy (Figure 7). Three loc i (PGM-I , PGM-2 and PGM-3) were p o s tu la t ed to be encoding PGM subun i ts in T. avetieus-. Only PGM-I and PGM-2 were expressed in muscle t i s s u e . The f a i n t s t a i n i n g o f the PGM-3 in l i v e r t i s s u e and i t s absence in muscle t i s s u e i s c o n s i s t e n t with the f ind ings o f o th e r au thors (Roberts e t a t . 1969, Hopkinson and Harr is 1968). PGM-I and PGM-3 were monomorphic in a l l popula t ions s t u d i e d , rep resen ted by s in g l e i n v a r i a n t bands. PGM-2 was polymorphic in the 51 + A PGM-5 PGM-2 PGM-I Figure 7. Phosphoglucomutase (PGM). The products o f th ree PGM lo c i appear to be p resen t in l iv e r samples. PGM-I , PGM-2 and PGM-3. PGM2 is polymorphic, PGM-I and PGM3 are monomorphic. 52 Donnelly River popu la t ion , but was monomorphic in the o th e r popula­ t ion s surveyed. Two a l l e l e s , PGM-2^'^ and PGM-2 ^ , were p re sen t in the Donnelly popu la t ion . Homozygous i n d iv idua l s f o r e i t h e r a l l e l e were rep resen ted by a s in g l e band while heterozygous i n d i ­ v idua ls e xh ib i t ed two bands o f a c t i v i t y (F igure 8) which i s t yp ic a l o f a monomeric p ro t e in . The p a t t e r n s found a re s im i l a r t o those repor ted in o th e r salmonids (noted above). The ex i s t en ce o f the polymorphism a t the PGM-2 locus c l a r i f i e s the i n t e r p r e t a t i o n of zymogram p a t t e r n s and s t r eng thens the v a l i d i t y o f the th r e e loc i p o s tu l a t e . I s o c i t r a t e dehydrogenase ( supe rna tan t form) - The supe rna tan t form o f i s o c i t r a t e dehydrogenase (IDHg) behaves e l e c t r o p h o r e t i c a l Iy as a d imeric molecule (Henderson 1968, Darnall and Klotz 1972). A l l e l i c v a r i a t i o n o f IDHg isozymes has been desc r ibed in a v a r i e t y o f salmonid spec ie s (A l lendorf at . 1975, May e t a t . 1975, Al len- do r f T973, Ropers e t a t . 1973, Engel e t a t . 1975, Wolf e t a t . 1970) which confirm the d imeric s t r u c t u r e in salmonid f i s h . The mode of inhe r i t ance of IDHg was i n i t i a l l y proposed to be te t r a som ic in rainbow t r o u t (Wolf e t a t . 1970) on the ba s i s o f phenotypic express ion a lone. A l lendo r f (1973) and A l lendorf and U t te r (1973) desc r ibed a system o f IDHg isozymes i d e n t i c a l to t h a t found by Wolf e t a t . . (1970). Based on the number o f bands and r e l a t i v e dosage, a two lo cu s , four 53 + 1.25 1.00 PGM-I 1 2 3 4 5 Figure 8. Phosphoglucomutase (PGM). PGM-I, c a th od a l, is mono- mo roh ic . PGM2 is polymorphic. Column 2 and 3 (1 .00/1 .00) homozygotes. “Column I and 4 (1 .00/1 .25) heterozygotes. Column 3 (1 .25/1 .25) homozygote. 54 a l l e l e system was proposed t o be encoding IDHg in these in d iv id u a l s . Breeding experiments v e r i f i e d t h a t in the s tock o f rainbow t r o u t examined by these a u tho r s , IDHg followed a disomic mode o f in h e r i t a n c e with the locus having been dup l i c a ted . Subsequent to th e se f i n d i n g s , breeding experiments were performed with the s tock examined by Wolf e t a l . (1970) which conc lu s ive ly v e r i f i e d t h a t , indeed , IDHg followed a disomic mode of in h e r i t a n c e (Engel e t a l . 1975). Liver t i s s u e e x t r a c t s o f T. arcticus were examined f o r IDHg a c t i v i t y and revealed the system dep ic ted in Figure 9. The IDHg pa t t e rn s were polymorphic in th r e e o f the popu la t ions surveyed. The express ion o f only th re e phenotypes in any one popu la t ion i s i n d i c a ­ t i v e o f a s in g l e disomic locus with a common and a v a r i a n t a l l e l e . This hypothes is o f a s i n g l e disomic locus f o r T. avcticus i s in c o n t r a s t t o the two disomic loc i po s tu la t ed f o r rainbow t r o u t (Al lendorf and U t te r 1973, Engel e t a l . 1975). The IDHg locus does not appear to have undergone gene d up l i c a t ion as in rainbow t r o u t (A l lendorf e t a l . 1975). A s in g l e disomic locus encoding IDHg has been repo r ted in chum salmon {Oneorhynchus keta) and confirmed through breeding experiments (May e t a l . 1975). A l l e l i c v a r i a t i o n was observed a t the IDHg locus in the Donnelly River, Wolf Lake, and Grebe Lake popu la t ions . The common a l l e l e des igna ted IDH encoded subun i ts which were e l e c t r o p h o r e t i c a l l y 55 Figure 9. I s o c i t r a te Dehydrogenase (IDH) is polymorphic. Three populations have an IDH v a rian t and one a l l e le common to a l l popu la tions. Column I (1 .25/1 .00) heterozygote. Column 2 3, 4, 5, 7 (1 .00/1 .00) homozygotes. Column 6 (1 .50/1 .00) heterozygote. Column 8 (1 .25/1 .25) homozygote. Column 9 (1 .50/1 .50) homozygote. Samples I , 2, 3, 8 are from the Donnelly r iv e r population . Samples 4 , 5, 6 , 7, 9 from the Grebe lake population . f56 I 25id en t i c a l in a l l popu la t ions . A v a r i a n t a l l e l e , des igna ted IDH5 " , was unique to the Donnelly River popu la t ion . A homozygous ind iv idua l f o r the v a r i a n t a l l e l e (1 .25 /1 :25 ) expressed a s i n g l e band o f a c t i v i t y with a g r e a t e r migration d i s t a n ce than a homozygous ind iv idua l f o r the common a l l e l e ( I .00 /1 .00 ) . A heterozygous ind iv idua l (1 .00 /1 .25 ) exh ib i ted a th r e e banded p a t t e r n with symmetrical s t a i n i n g i n t e n s i t y , c h a r a c t e r i s t i c of a d imeric molecule. I 50Another v a r i a n t a l l e l e , IDH5 " , was unique to the two Yellow­ stone Park popu la t ions . The isozyme o f an ind iv idua l homozygous fo r t h i s a l l e l e had a c h a r a c t e r i s t i c migra tion d i s t a nce g r e a t e r than e i t h e r the 1 .00 /1 .00 in d iv idua l s or the 1 .25 /1 .25 i n d iv id u a l s . A heterozygous ind iv idua l f o r t h i s v a r i a n t (1 .00 /1 .50 ) again expressed a th ree banded phenotype. As noted in Figure 9 a 1 .00 /1 .50 ind iv idua l has an asymmetrical s t a i n i n g i n t e n s i t y which may r e f l e c t a d i f f e r ­ e n t i a l a c t i v a t i o n o f the two a l l e l e s o r d i f f e r e n t i a l a c t i v i t y o f these a l l e l e p roducts . Only 1 .00 /1 .00 ind iv idua l s were found in the Fuse Lake popula­ t i o n which may again r e f l e c t a founder e f f e c t . T r an s fe r r in - (Tfn) i s a monomeric B-g lobulin which e x h ib i t s a high degree o f polymorphism in most v e r t e b r a t e spec ie s (Manwell and Baker 1970). The ex is t ence o f an ex tens ive amount o f g ene t i c v a r i a ­ t ion has been repor ted in t e l e o s t s , with more than t h i r t y spec ie s 57 being polymorphic (reviewed by Kirpichnikov 1973, DeLigny 1969). The majo r i ty o f these polymorphisms r e f l e c t codominant i n h e r i t a n c e of a l l e l e s a s so c ia t ed with a s in g l e locus (Kirpichnikov 1973). I n h e r i ­ tance s tu d i e s v e r i f i e d the i n t e r p r e t a t i o n o f one polymorphic disomic locus (Valenta e t a l . 1976, U t t e r e t a l . 1973). The la rg e amount o f v a r i a b i l i t y o f t r a n s f e r r i n i s well documented in salmonid spec ie s (Hershberger 1970, Wright and Atherton 1970, Moller 1970, U t t e r e t a l . 1970, U t t e r and Hodgins 1972, Eckroat 1973, A l lendo r f 1973, U t t e r e t a l . 1973). In a l l cases th e re i s a simple a d d i t i v e p a t t e rn in he terozygotes f o r a t r a n s f e r r i n polymorphism. The presence o f th re e o r more a l l e l e s has been observed in rainbow t r o u t and var ious salmon spec ie s (U t te r e t a l . 1970, 1973, U t t e r and Hodgins 1972, Reichenbach-Klinke 1973) and a l so in brook t r o u t (Eckroat 1973). Serum samples o f ind iv idua l g ray l ing were analyzed fo r t r a n s ­ f e r r i n phenotypes. The t r a n s f e r r i n bands (F igure 10) were i d e n t i f i e d as the bands having an e l e c t r o p h o r e t i c mob i l i ty s im i l a r in p o s i t i o n , to those o f rainbow t r o u t and salmon (U t te r e t a l . 1970, 1973, Reichenbach-Klinke 1973). I t i s assumed t h a t these bands in T. apotieus a re a l so t r a n s f e r r i n s because o f t h i s e l e c t r o p h o r e t i c i d e n t i t y and the taxonomic r e l a t i o n s h i p o f th e se s p ec ie s . T ran s fe r r in was found to be polymorphic in the Donnelly R ive r , Wolf Lake, and Grebe Lake popu la t ions . On the b a s i s o f observed > + 58 Figure 10. T ran sfe rrin polymorphism (T fn). Two a l le le s were found in the Donnelly r iv e r population and th ree in the Yellowstone Park popu la tions. Columns I , 2, 3, 4, 5 7, 8, 9, 11, 12 (1 .00/1 .00) homozygotes. Column 6 (1 .20/1 .20) homozygote. Column 10 (1 .10/1 .00) h e te ro ­ zygote. Column 1 , 3 , 7 Donnelly r iv e r . A ll o thers from Grebe lake. 59 phenotypes and the monomeric s t r u c t u r e o f T fn , th r e e codominant a l l e l e s ( T f n ^ T f n ^ ' and T f n ^ in o rde r o f i n c re a s ing anodal migration) a t a s ing le disomic locus were p o s tu la t ed to encode Tfn in the two Yellowstone Park p opu la t i o n s . Six phenotypes of e i t h e r one o r two o f t h r e e d i f f e r e n t bands a re expected to r e s u l t from the codominant express ion o f th r e e a l l e l e s . The s i x phenotypes a cco r ­ d ingly a re des igna ted 1 .00 /1 .00 , 1 .00 /1 .10 , I .00 /1 .20 , I .10 /1 .10 , 1 .10 /1 .20 and 1 .20 /1 .20 . Four o f these phenotypes a re shown in Figure 10. The only phenotype not found in the in d iv id u a l s sampled was I .10 /1 .20 . The lack o f obse rva t ion o f t h i s phenotype i s most l i k e ly the r e s u l t o f the small sample s i z e r e s u l t i n g in no d e te c t io n of I .10 /1 .20 whose maximum expected value i s .013. E l e c t r o p h o r e t i c a l l y i d e n t i c a l a l l e l e s to T f n ^ and Tfn^ I 20were expressed in the Donnelly River popu la t ion , but the Tfn * a l l e l e was absen t . The th re e expected phenotypes (1 .00 /1 .00 , 1 .00 / 1.10 and 1 .10 /1 .10) r e f l e c t i n g two codominant a l l e l e s were observed. The common a l l e l e Tfn^’^ was the only one expressed in the Fuse Lake popu la t ion , thus only the common phenotype (1 .00 /1 .00 ) was observed. The lack o f v a r i a t i o n a t the Tfn locus in t h i s popula t ion i s presumably due to a founder e f f e c t . Glucose and hexose 6-phosphate dehydrogenase - Two major e l e c t r o - p h o r e t i c a l l y d i s t i n c t components o f glucose 6-phosphate dehydrogenase 60 a c t i v i t y a re found in v e r t e b r a t e organisms. One i s h igh ly s p e c i f i c f o r the u t i l i z a t i o n o f g lucose 6-phosphate with NADP as s u b s t r a t e and coenzyme, r e s p ec t iv e ly (Noltman and Kuby 1963). The second form is d i s t in gu i sh ed by i t s a b i l i t y , t o ca ta ly ze the ox ida t ion o f glucose 6-phosphate , ga lac to se 6-phosphate , 2-deoxyglucose 6-phosphate and glucose as s u b s t r a t e s , with e i t h e r NAD o r NADP func t ion ing as the coenzyme (Shaw 1966, Ohno e t a t . 1966, Beu t le r and Morrison 1967, Shaw and Koen 1968). The isozyme with g lucose 6-phosphate s p e c i f i c dehydrogenase a c t i v i t y i s des igna ted glucose 6-phosphate dehydrogen­ ase (G6PD), whi le the enzyme with the b roader range o f s u b s t r a t e s p e c i f i c i t y i s des igna ted hexose 6-phosphate dehydrogenase (H6PD) f o r convenience o f d i s t i n c t i o n (Shaw 1966, Ohno e t a l . 1966, Shaw and Koen 1968). Ver teb ra te G6PD and H6PD isozymes have been shown to be encoded by d i s t i n c t gene l o c i . G6PD i s sex l inked in mammals (Kirkman and Hendrickson 1963, Richardson e t a l . 1971) but i s under autosomal gene con tro l in b i rd s and f i s h (Manwell and Baker 1969, Yamauchi and Goldberg 1973). G6PD i s lo c a l i z e d in the nuc lea r and cytoplasmic f r a c t io n s o f th e c e l l (Beu t le r and Morrison 1967). In c o n t r a s t , H6PD is autosomally i n h e r i t e d in mammals (Shaw 1966, Ohno e t a l . 1966, Shaw and Koen 1968) and i s l o c a l i z ed in the microsomal f r a c t i o n o f the c e l l (Beu t le r and Morrison 1967, Metzger e t a l . 1965). 61 , G6PD e x i s t s as two c a t a l y t i c a l l y a c t i v e forms, dimer and t e t r ame r (Bonsignore e t a l . 1970)» being mostly t e t r am e r i c in some organisms (Yamauchi and Goldberg 1973) but dimeric in o th e r s (Shaw and Koen 1968). In c o n t r a s t H6PD e x h ib i t s a dimeric s t r u c t u r e as evidenced in zymograms o f a l l e l i c v a r i a n t s (Stegeman and Goldberg 1971). The t i s s u e d i s t r i b u t i o n of G6PD appears ub iqu i tous in v e r t e b r a t e c e l l s (B eu t le r and Morrison 1967). H6PD i s most a c t i v e in t h e l i v e r and kidney (Beu t le r and Morrison 1967) with a c t i v i t y in o th e r t i s s u e s only f i v e to ten pe rcen t o f t h a t in l i v e r e x t r a c t s (Stegeman and Goldberg 1971). The occurrence o f both G6PD and H6PD in the l i v e r s o f salmonids has been c l e a r l y demonstrated by var ious au thors (Stegeman and Goldberg 1971, Shatton e t a l . 1971s Ohno e t a l . 1966). The exac t number o f isozymes in the multi -banded zymograms o f rainbow t r o u t i s , however, no t known. Stegeman and Goldberg (1971, 1972) demon­ s t r a t e d t h a t in the genus Sa lve linu s G6PD appears to be t e t r am e r i c ( exh ib i t ing f i v e bands) with H6PD e x i s t i n g as a d imeric molecule. The ex is t ence o f two codominant gene loc i with d i f f e r e n t a l l e l e s has been po s tu la t ed to be determin ing the G6PD bands in t r o u t (Ohno .1966, Yamauchi and Goldberg 1973, Stegeman and Goldberg 1971). However, Cederbaum and Yoshida (1975) r e c en t l y proposed t h a t G6PD in rainbow t r o u t l i v e r may be encoded by a s ing le gene lo cu s , two a l l e l e system. 62 with p o s t t r a n s l a t i o n a l modif ica t ion o f the enzyme accounting f o r the complex banding p a t t e r n s . On the ba s i s o f a l l e l i c v a r i a t i o n s tu d i e s by Stegeman and Goldberg (1971, 1972), H6PD i s be l ieved to be the product o f a s in g l e autosomal gene locus . E lec t ropho re t i c a n a ly s i s o f G6PD a c t i v i t y in l i v e r t i s s u e e x t r a c t s o f T. a ro tious revea led t h a t t h i s t i s s u e conta ined four d i s t i n c t zones o f a c t i v i t y when glucose 6-phosphate was used as sub­ s t r a t e in s t a i n i n g (F igures 11 and 13). The band appearing second from the cathodal end ( l i v e r samples) was i d e n t i f i e d as H6PD, e x h ib i t i n g broad s u b s t r a t e s p e c i f i c i t y . i n d i c a t e d by s t a i n i n g with ga lac tose 6-phosphate and glucose with NAD o r NADP as the coenzyme (Figure 11). The re fo re , both forms o f G6PD a re p re sen t in T h aro tious as in o the r v e r t e b r a t e s . Three zones o f G6PD a c t i v i t y , each rep re sen ted by a s i n g l e band, i s the common phenotype. The middle zone o f G6PD a c t i v i t y i s g r e a t e r in s t a i n in g i n t e n s i t y than e i t h e r o f the o th e r two zones (F igure 11, e ry th ro c y te s , and Figure 13). This phenotypic p a t t e r n i s sugges t ive o f two loc i with a l l e l e s encoding subun i ts o f d i f f e r e n t e l e c t r o ­ pho re t ic mob i l i ty f o r a dimeric molecule. The two loci a re d e s ig ­ nated G6PD-2 and G6PD-3 in o rde r o f i n c re a s ing anodal m ig ra t ion . A v e r i f i c a t i o n o f t h i s model o f in h e r i t a n c e was ob ta ined by f ind ing ind iv idua ls in the Grebe and Wolf Lake popula t ions possess ing e l e c t r o ­ pho re t ic p a t t e r n s as shown in lane two and fou r o f Figure 12. These. 63 + -G6PD-3 H6PD -G6PD-2 -G6PD-1 1 2 3 4 5 6 7 Figure 11. Glucose-6-Phosphate Dehydrogenase (G6PD) and Hexose-6- Phosphate Dehydrogenase (H6PD) expression in ery th rocy tes and eye t is s u e . Liver t is su e samples have the same banding p a tte rn as e ry th ro cy tes . Column I , 2, 3, 6 and 7 are eye samples from ind iv idual f is h . Column 4 and 5 ery th rocy tes from ind iv idual f is h . 64 1 2 3 4 Figure 12. Glucose-6 -Phosphate Dehydrogenase-5 (G6PD-3) is poly­ morphic in the Yellowstone Park popu la tions. Column I mixture o f homogenates app lied to column 2 and 4. Column 2 G6PD-3 (1 .00/1 .00) homozygote. Column 3 G6PD-3 (1 .00/1 .10) heterozygote. Column 4 G6PD-3 (1 .10/1 .10) homozygote. Liver samples. 65 1 2 5 4 5 6 7 8 9 10 11 12 Figure 13. Hexose-6- Phosphate Dehydrogenase (H6PD) polymorphism. Donnelly River samples have a s l ig h t ly f a s te r anodal m igrating form than the Grebe Lake (YNP) samples. Columns I , 2, 3, 7, 8 and 9 (1 .10/1 .10) homozygotes from Donnelly R iver. Columns 4, 5, 6, 10, 11 and 12 (1.00/1.00) homozygotes from Grebe Lake. Liver homogenates. 66 p a t t e r n s i n d i c a t e th e presence o f a v a r i a n t a l l e l e a t the G6PD-3 locus . A homozygous ind iv idua l f o r the v a r i a n t a l l e l e (des ignated G6PD-3^ '^ ) i s r ep resen ted by a s in g l e band with a g r e a t e r e l e c t r o ­ pho re t i c mob i l i ty than an ind iv idua l homozygous f o r the common a l l e l e (G6PD-3^*^). Heterozygotes (1 .00 /1 .10 ) e x h ib i t a th ree banded phenotype c h a r a c t e r i s t i c of th e d imeric s t r u c t u r e with a band in te rmed ia te between th e two homozygote bands. The var ious isozyme p a t t e r n s a re dep ic ted in Figure 12. The presence o f t h i s polymorphism prov ides add i t i o n a l evidence to support the two locus p o s tu l a t e . Since a he te rodimer i s formed of subun i ts from the two l o c i , a band o f g r e a t e r e l e c t r o p h o r e t i c mob i l i ty in the h e t e r o d imeric region would be expected in a homo­ zygous ind iv idua l f o r the v a r i a n t a l l e l e a t the G6PD-3 locus . Furthermore, an ind iv idua l heterozygous f o r the v a r i a n t G6PD-3 a l l e l e would have two he te rod imer ic bands, formed between subuni ts , o f the G6PD-2 and the two G6PD-3 a l l e l e s . Figure 12 c l e a r l y shows t h i s to be the case . These r e s u l t s a re found whether NADP i s used in the g r ind ing so lu t io n o r no t , a l though the presence o f NADP does enhance r e s o lu t i o n . The data i s in agreement with the two codominant lo c i p o s tu la t ed by Ohno e t a t . (1966) f o r rainbow t r o u t and Stegeman and Goldberg (1971) f o r brook t r o u t . The r e s o lu t ion ob ta ined in th e se zymograms o f T,. a rc tieu s i s much more convincing than those 67 used by Cederbaum and Yoshida (1975) in p o s tu l a t i n g the presence o f a s i n g l e locus . Analysis o f eye t i s s u e and red blood c e l l s r e s u l t e d in the p a r a l l e l express ion o f the two G6PD, loc i with the h e t e r o d imeric region again e v id en t (F igure 11). The lack o f H6PD a c t i v i t y in the eye t i s s u e c l a r i f i e s i n t e r p r e t a t i o n o f l i v e r zymograms s ince one can assume t h a t none o f the a c t i v i t y in t h i s region i s due to G6PD. An add i t io n a l band c lo se to the o r i g i n was expressed in eye t i s s u e and red blood c e l l s , zymograms (F igure 11). No d i s t i n c t he te rod imer ic reg ion was formed between t h i s band and the subun i ts o f G6PD-2 o r G6PD-3. The band may r e p r e s en t a d i s s o c i a t i o n product o f the G6PD-2 but i t s c o n s i s t e n t presence in a l l samples in equal i n t e n s i t y and absence in l i v e r sugges ts an add i t iona l l o c u s , d e s ig ­ nated G6PD-1, which i s a c t i v e in these t i s s u e s but not in the l i v e r . In summary, the G1SPD-I and G6PD-2 loc i were monomorphic in a l l popu la t ions . The G6PD-3 locus was monomorphic in the Donnelly and Fuse popu la t ion s , but was polymorphic in the Grebe and Wolf Lake popu la t ions . A region o f HSPD a c t i v i t y was expressed in a l l popula t ions surveyed. As noted in Figure 13, HSPD a c t i v i t y i s rep re sen ted by an a rea o f d i f f u s e s t a i n i n g . I t i s po s tu la t ed t h a t a s i n g l e locus i s r ep re sen ted by t h i s HSPD a c t i v i t y , a l though i t appears two bands 68 a re p re s en t a t t imes . As shown in Figure 13, the H6PD band in Donnelly River and Fuse Lake , ind iv idua ls has a c h a r a c t e r i s t i c migra­ t i o n d i s t a n ce g r e a t e r than the in d iv id u a l s from t h e Yellowstone popu la t ions . A mixture o f an ind iv idua l sample from each popula tion r e s u l t s in an a rea s t a i n i n g which i s c l e a r l y a d d i t i v e o f the areas s t a i n ed in ind iv idua l samples. The r e s u l t s provide evidence t h a t th e H6PD locus i s f ix ed f o r a l l e l e s which code fo r subun i t s of d i f f e r e n t e l e c t r o p h o r e t i c mob i l i ty . The a l l e l e in th e Grebe and Wolf Lake popula t ions i s des igna ted H6PD1‘00 and t h a t in the Donnelly I TORiver and Fuse Lake popula t ions i s H6PD * . No in t r apopu la t ion a l v a r i a t i o n was found to e x i s t . Malic enzyme (NADP - MDH) - Malic enzyme o therwise known as NADP dependent malate dehydrogenase e x i s t s in mammalian t i s s u e in two forms, supe rna tan t and mitochondria l (Henderson 1966, Shows e t d l 1970). Evidence from e l e c t r o p h o r e t i c s tu d i e s o f the mouse sugges t a t e t r am e r i c s t r u c t u r e f o r both the supe rna tan t (MEg) and mitochon­ d r i a l (MEffl) form o f t h i s enzyme (Shows and Ruddle 1968, Baker and Mintz 1969, Povey e t a l . 1975). The mitochondria l form migra tes l e s s anodal ly than the so lub le form in some spec ie s (Cohen and Omen 1972) but may move a g r e a t e r d i s t a n ce in o th e r s (Henderson 1966). Two autosomal gene loc i (MEg and MEm) determine the so lub le and mitochondria l forms, r e s p e c t i v e ly (Cohen and Omen 1972, Povey e t a l . 1975). 69 The ME systems have not been as well s tud ied in t e l e o s t s . The lack o f v a r i a t i o n in spec ie s s tud ied has not al lowed de te rmina t ion o f the molecular s t r u c t u r e in t e l e o s t f i s h (U t te r e t a l . 1973, A l lendorf 1973, K r i s t i an sson and McIntyre 1973). Frydenberg and Simonsen (1973) , on the b a s i s o f e l e c t r o p h o r e t i c p a t t e r n s , sugges ted t h a t a s impler molecule e x i s t e d in the t e l e o s t , Zparoes, than the te t r ame r found in the house mouse (Shows and Ruddle 1968, Povey e t a l., 1975). They considered the enzyme in Zoareee to be c o n t ro l l e d by a s ing le locus . A l lendo r f e t a l . (1975) have repo r ted a d i f f e r e n t band of a c t i v i t y p re sen t in muscle than in l i v e r in rainbow t r o u t , but the exac t number o f lo c i was not determined. Liver and muscle t i s s u e e x t r a c t s were surveyed fo r ME a c t i v i t y in T. a re tie u s . An id e n t i c a l phenotypic p a t t e rn (F igure 14) was expressed in both t i s s u e s . A group o f f i v e bands which migrates f u r t h e r than any MDH isozymes was i d e n t i f i e d as the supe rna tan t form: o f ME (MEg). . A s lower d i f f u s e s t a i n i n g band with a migra t ion d i s ­ tance in te rmed ia te between MDH5 and MDHm isozymes was i d e n t i f i e d as the mitochondria l form o f ME (ME ).m The presence o f f i v e bands in every ind iv idua l i n d i c a t e s the presence o f two loc i with a l l e l e s encoding subuni ts of d i f f e r e n t e l e c t r o p h o r e t i c mob i l i ty f o r a t e t r am e r i c molecule. The two loci were des igna ted ME5-I and ME5 -Z. The i n t e n s i t y o f the ind iv idua l 70 4- Figure 14. Malic enzyme (M.E.) . The supernatant form of M.E. appears to have a multimeric s tru c tu re perhaps produced by lo c i which are d i f f e r e n t ia l ly a c tiv e . L iver homo­ genates. The m itochordria l form appears to be mono- morphic. 71 bands var ied with the ind iv idua l sample sugges t ing t h a t one o f the loc i may be polymorphic whi le th e o th e r i s monomorphic. A r a r e v a r i a n t was a l so i d e n t i f i e d in one ind iv idua l in the Grebe Lake popu la t ion . Since v a r i a t i o n i s suspec ted but the number o f a l l e l e s i s no t known, th e se loc i were excluded from the q u a n t i t a t i v e c a lcu ­ l a t i o n s . However, the presence o f two loc i coding f o r a t e t r am e r ic molecule i s sugges ted . The s t r u c t u r e i s s im i l a r to t h a t repor ted in o th e r v e r t e b r a t e s (d iscussed p r e v io u s ly ) . The ME5 locus appears to be dup l ic a ted in T. a ro tious . The MEm band was e l e c t r o p h o r e t i c a l l y i d e n t i c a l in a l l popu la t ions . Since no v a r i a t i o n i s p r e s en t , noth ing can be deduced about the s t r u c ­ t u r e o f t h i s molecule. One s t r u c t u r a l gene probably codes f o r t h i s enzyme. Serum p ro te in s - T e leo s t spec ie s have been shown to possess i n t r a s p e c i e s s p e c i f i c i t y o f plasma p ro t e in p a t t e r n s (Woods and Engle 1957, Tsuyki and Roberts 1966). Although f i s h serum p ro t e in s a re as y e t impe r fec t ly s tud ied from a gene t i c po in t o f view (Kirpichnikov 1973), they have been used s u c c e s s f u l l y in work on i n t r a s p e c i f i c sy s tema t ic s (Lukjanenko and Popov 1969, Wright and Hasle r 1967, R e in i tz 1973, Booke 1964, DeLigny 1969). In analyz ing serum p ro te in s f o r gene t i c v a r i a t i o n , i t must be noted t h a t serum p ro t e in p a t t e rn s reso lved e l e c t r o p h o r e t i c a l l y vary with phys io log ica l and environmenta l 72 cond i t ions (Booke 1964, Thurston 1967). A p r in c ip a l v a r i a t i o n noted i s r e l a t e d to sex and ma tu r i ty in females (reviewed by DeLigny 1969, Feeney and Brown 1974). The non-gene t ic v a r i a t i o n of serum p ro te in s appears to involve q u a n t i t a t i v e changes r a t h e r than the presence o r absence o f bands (Booke 1964, Thurston 1967, Feeney and Brown 1974, DeLigny 1969). As DeLigny (1969) sugges t s , in any study o f v a r i a t i o n in n o n - i d e n t i f i e d components o f serum p r o t e i n s , s u f f i ­ c i e n t popula t ion da ta and ana ly s i s o f the composition o f the samples with regard to sex , m a tu r i t y , and development s tage appears an ab so lu t e requirement. Furthermore, a good r e so lu t io n o f the p a t t e rn which allows a sharp d i s t i n c t i o n between ind iv idua l f r a c t i o n s i s needed in o rde r to avoid wrong i n t e r p r e t a t i o n o f the observed v a r i a t i o n . The plasma p ro t e in s o f f i s h as y e t do not appear t o have accura te c l a s s i f i c a t i o n . No conclus ions as to s im i l a r i t y to human plasma f r a c t i o n s can be made (Feeney and Brown 1974). Recent a t tempts a t c l a s s i f i c a t i o n o f the var ious f r a c t i o n s o f plasma p ro t e in s o f various salmonid spec ie s have been done ( P e r r i e r e t a t . 1973, Reichenbach- Klinke 1973). However, i t was beyond the scope o f the p r e s en t study to a ttempt to i d e n t i f y and biochemica l ly c h a r a c t e r i z e th e various f r a c t i o n s o f serum p ro t e in s observed on s t a r ch ge ls o f Thymallus avo tio u s . 73 Serum samples o f the fou r popu la t ions o f Thymallus avotious under i n v e s t i g a t i o n were sub jec ted to e l e c t r o p ho r e s i s and s t a in ed with a general p ro t e in s t a i n . Electropherograms o f th e serum p ro te in s a re shown in Figures 14 and 15. Samples were normally f rozen before e l e c t r o p h o r e s i s but subsequent s tu d i e s revea led t h a t f r e e z ing and thawing had no e f f e c t on the e l e c t r o p h o r e t i c p a t t e r n s o f the serum p ro t e i n s . Electropherograms o f serum samples revea led 6 zones of s t a i n i n g as shown diagrammatica l! . / in Figure 16. Zone 2 was i d e n t i ­ f i e d as t r a n s f e r r i n which has been d e a l t with p rev ious ly . The only o th e r zone adequate ly reso lved was Zone 5. The r e s o lu t i o n of t h i s zone was e x c e l l e n t and the number o f bands could be determined p re ­ c i s e l y . Ind iv idua ls e xh ib i t e d from th r e e to f i v e bands in t h i s reg ion . A t o t a l o f twelve d i f f e r e n t e l e c t r o p h o r e t i c phenotypes were observed as dep ic ted in Figure 17. Fu r the r ana ly s i s revea led t h a t the zone could be f u r t h e r d iv ided in to two groups des igna ted A (most a noda l ) , B ( l e a s t anoda l ) . Group A was found to be r ep re sen ted by two in ten se s t a i n i n g bands in a l l i n d iv id u a l s in both the Grebe Lake and Wolf Lake popu la t ions whose express ion appeared independent of the B group (F igure 17). I t was p o s tu l a t ed t h a t th e se bands r ep re ­ sen ted two gene l o c i , SP-2 and SP-3, in o rde r of in c re a s ing anodal m ig ra t ion , encoding p ro te in s o f d i f f e r e n t e l e c t r o p h o r e t i c mob i l i ty . Ind iv idua ls from the Donnelly River popu la t ion , however, e xh ib i t e d e i t h e r two o r th r e e bands in t h i s reg ion . One band o f 74 Figure 15. Electropherograms of serum p ro te n s . a . Phenotypes o f the Yellowstone Park popu la tions. All ind iv idua ls are homozygous a t the SP-2 ( I .0 0 /1 .OOf locus and the SP-3 (1 .00/1 .00) locus. The ind iv iduals are polymorphic a t the SP-I locus: Column I - homo­ zygote (1 .10 /1 .10 ); column 2 and 4 - homozygote (1 .00 /1 .00 ); column 3, 5 and 6 - heterozygotes (1 . 00/ 1 . 10) . b. Phenotypes o f Donnelly River in d iv idua ls . All ind iv idua ls are homozygous (1.00/1.00) a t the SP-% locus. Ind iv iduals are polymorphic a t th e SP-I and SP-2 l o c i : Columns I and 3 - SP-I (1 .10 /1 .10 ), SP-2 (1 .10 /1 .20 ); column 2 - SP-I (1 .10 /1 .00 ), SP-2 (1 .10 /1 .10 ); column 5 - SP-I (1 .10 /1 .00 ), SP-2 (1 .10 /1 .20 ); column 4 - Yellowstone Park ind iv idual - SP-I (1 .00 /1 .00 ), SP-2 (1 .00 /1 .00 ), SP-3 (1 .00 /1 .00). 75 4 - I----SP-3 — I i— SP-2 I -SP -I ( 1 . 10) —'---- SP-I (1.00) I 2 3 4 5 6 + -----SP-2 (1.20 r— SP-3 ^ i—SP-2 (1 .10) STAEMAN OFPRRI 1----SP-I (1.10) -----SP-I (1.00) 1 2 3 4 5 76 :z= z Zone I Zone 2 Zone 3 Zone 4 — Zone 5 Zone 6 Figure 16. Group ■■ C % 3 C .Z 3 I S I SP -21*20 era era era SP-21 ' 10 Group B C Z 3 m m c . z 3 niiUI era — SP-21 ' 00 S P - I1 *10 t t a « 9 e ra i V era n n U S P - I1 -00 Figure 17. The tw elve observed phenotypic pa tterns of electropherogram s of grayling serum proteins in Zone 5. Columns 1 -9 , Donnelly River; Columns 10-12 , Yellowstone Park popu la tions. The genotypes of th e ind iv iduals are: I - SP-I (1 .0 0 /1 .0 0 ) , SP -2 (1 .1 0 /1 .1 0 ) ; 2 - S P -K l . 0 0 / 1 . 10 ),SP -2 (1 .1 0 /1 .1 0 ) ; 3 -SP -l ( 1 .1 0 /1 .1 0 ) , SP-2 (1.1 0 /1 .1 0 ) ; 4 - SP - I (1 .00 /1 .00 ), SP-2 (1 .2 0 /1 .2 0 ) ; 5- SP-I (1 .0 0 /1 .1 0 ) , SP-2 (1 .2 0 /1 .2 0 ) ; 6 -SP -l (1 .1 0 /1 .1 0 ) , S P -2 (1 .2 0 /1 .20) ; 7 - SP-I (1 .0 0 /1 .0 0 ) , SP-2 (1 .1 0 /1 .2 0 ) ; 8 - SP-I (I . 0 0 /1 .1 0 ) , SP-2 (1 .1 0 /1 .2 0 ) ; 9 - SP-I (1 .1 0 /1 .1 0 ) , SP-2 (1.1 0 /1 .2 0 ) ; 10- SP-I (1.0 0 /1 .0 0 ) , SP-2 (1 .0 0 /1 .0 0 ) ; 11- SP-I (1 .0 0 /1 .1 0 ) , SP-2 (1.0 0 /1 .0 0 ) ; 12- SP-I ( l . 1 0 /1 .1 0 ) , SP-2 (1.0 0 /1 .0 0 ) . All ind iv iduals are homo­ zygous for the common a lle le (1 .0 0 /1 .0 0 ) at the SP-3 lo cu s . s t rong i n t e n s i t y was c o n s i s t e n t in a l l samples, and was e l e c t r o - p h o r e t i c a l l y i d e n t i c a l to the SP-3 band o f the Yellowstone popula­ t io n s (F igure 17), and was des igna ted as such. Two o t h e r bands were a l so observed in t h i s reg ion , one which had a s l i g h t l y f a s t e r migra­ t i o n d i s t a nce than the SP-3 band and one which had a s l i g h t l y slower migra t ion d i s t a n c e , in te rmed ia te between the SP-3 and the SP-2 band o f the Yellowstone ind iv id u a l s . These bands a re cons idered to be the equ iva l en t o f the bands produced by the SP-2 locus in the Yellow­ stone popu la t ion s , s ince no band equ iva l en t in migra t ion d i s tance to the SP-2 band o f the Yellowstone popula t ions i s found in the Donnelly popu la t ion . Ind iv idua ls e xh ib i t e d e i t h e r an in t en se s t a i n in g f a s t band, an in ten se s t a i n in g slow band, o r had both bands p resen t in weaker i n t e n s i t y . These r e s u l t s lead to the i n t e r p r e t a t i o n o f a one lo cu s , two a l l e l e system encoding a monomeric p r o t e i n , with heterozygous ind iv id u a l s express ing two weaker s t a i n i n g bands, the m o b i l i t i e s o f which a re i d e n t i c a l t o th e bands expressed in both types o f homozygous i n d iv id u a l s . The a l l e l e common to the Yellow­ s tone popula t ions was des igna ted SP -2^ '00 , those in the Donnelly I TD I PD River popula t ion SP-2 and SP-2 in o rde r o f in c re a s ing anodal m igra t ion . On the b a s i s o f t h i s hypo the s i s , a l l e l e f r equenc ies were determined f o r the two a l l e l e s in the Donnelly River popu la t ion . In the Group B reg ion , ind iv idua l s i n a l l popu la t io n s , except the Fuse Lake popu la t ion , exh ib i ted e i t h e r one o r two bands of 78 79 a c t i v i t y (F igure 17). A s i n g l e lo cu s , two a l l e l e system encoding a monomeric p ro te in was po s tu la t ed to be r e spons ib le f o r t h i s p a t t e r n o f v a r i a t i o n , with homozygous i n d iv idua l s e x h ib i t i n g a s i n g l e band o f s t rong i n t e n s i t y and heterozygous ind iv idua l s having two bands o f weaker i n t e n s i t y . This locus was des igna ted SP-I with the two a l l e l e s termed SP-V and SP-V ’^ in o rde r o f i n c re a s ing anodal m igra t ion . Evidence in support o f t h i s g ene t i c i n t e r p r e t a t i o n l i e s in the express ion of the twelve expected phenotypes. Nine phenotypes would be expected to r e s u l t in the Donnelly River popu la t ion , and th re e in the Wolf and Grebe Lake popu la t ion s , as i s indeed the case . Fuse Lake ind iv idua l s expressed an i d e n t i c a l SP-3 band as t h a t found in both the Yellowstone Park and Donnelly River popu la t ions . The Fuse Lake popula t ion was no t polymorphic a t the SP-2 lo cu s , but I 10 appeared f ixed f o r the SP-2 ’ a l l e l e which was p re s en t in the Donnelly popu la t ion s , e x h ib i t i n g a band e l e c t r o p h o r e t i c a l Iy i d e n t i c a l to t h a t observed in the Donnely popu la t ion . At the SP-I locus the Fuse Lake popula t ion was f ixed f o r the I . TO a l l e l e with no apparent v a r i a t i o n . As p rev ious ly no ted , any s tudy o f v a r i a t i o n o f serum p ro te in p a t t e r n s must have s u f f i c i e n t popula t ion da ta to determine i f the v a r i a t i o n has a b a s i s o t h e r than g ene t i c . In the p re s en t c ase . 80 records were kept as to the sex , age and rep roduc t ive cond i t ion o f the ind iv idua l s sampled. No c o r r e l a t i o n e x i s t e d between these f a c to r s and the p a t t e r n exh ib i t e d by the ind iv idua l . Since the v a r i a t i o n observed f o r these serum p ro t e in s has not been adequate ly s tud ied in r e l a t e d s p e c i e s , the q u a n t i t a t i v e ana ly s i s o f these systems w i l l be d e a l t with s ep a r a t e ly . Q uan t i t a t i v e Analysis o f Genetic V a r i a b i l i t y A l l e l e f r equenc ie s o f t h i r t y - f i v e enzyme and p ro t e in loc i are p resen ted in Table 3, f o r the fou r popula t ions o f Thymallus avctlous surveyed. The t a b l e shows the number o f a l l e l e s d e tec ted by e l e c t r o ­ phores is a t each locus . The h e te rozygos i ty a t each locus i s a l so O c a l c u l a t e d . Homozygosity a t a locus ( j ) i s def ined as E x where x i s the frequency o f the i - t h a l l e l e . Heterozygosity f o r the p locus (h) i s def ined as I-E x (Nei 1975). The a l l e l e f requencies used a re those obta ined d i r e c t l y from the observed d a ta . The enzymes and p ro te in s surveyed in t h i s study were div ided in to t h r e e groups. Group I inc ludes those enzymes involved in g lu ­ cose metabolism. Group I I inc ludes non-glucose metabo l iz ing enzymes and Group I I I in c ludes non-enzymatic p r o t e i n s . The g ene t i c b a s i s o f isozyme polymorphisms scored on the gels were not v e r i f i e d d i r e c t l y by progeny s t u d i e s . The g ene t i c i n t e r ­ p r e t a t i o n o f the observed v a r i a t i o n in simple Medelian terms i s 81 Table 3. A l l e l e f r equencies and degree o f h e te rozygos i ty in 35 loci examined in fou r popula t ions o f Thymallus a ro ticu s . (h = he te rozygos i ty ) Locus A l le l e s Grebe Wolf DonnelIy Fuse Group I . Glucose Metabol iz ing Enzymes AGPD-I 1.00 1.00 1.00 1.00 1.00 (h) 0.0 0.0 0 .0 0.0 AGPD-2 1.00 1.00 1.00 1.00 1.00 (h) 0.0 0.0 0 .0 0.0 AGPD-3 1.00 1.00 1.00 1.00 1.00 (h) 0 .0 0.0 0 .0 0.0 Hexokinase-I 1.00 1.00 1.00 1 .00 1.00 (h) 0 .0 0.0 0 .0 0.0 H6PD-1 1.00 1.00 1.00 1.25 — — — — — — — — 1.00 1.00 ( h ) 0.0 0 .0 0 .0 0 .0 IDH-I 1.00 0.98 0.98 0.97 1.00 S 1.10 — — — — — — — — 0.03 — — — — 1.20 0.02 0.02 — — — — — — — — ( h ) 0.04 0.04 0.06 0 .0 IDH-I 1.00 1.00 1.00 1.00 1.00 ( h ) m 0.0 0.0 0 .0 0.0 IDH -2 1.00 1.00 1.00 1.00 1.00 L. Swed. J . Ag. Res. 5: 185-192. Kimura, M. and J . F. Crow. (1964) The number of a l l e l e s t h a t can be mainta ined in a f i n i t e popu la t ion . Genetics 49: 725-738. King, M. and A. C. Wilson. (1975) Evolution a t two l e v e l s : molecular s im i l a r i t i e s and b io log ic a l d i f f e r en ce s between humans and chimpanzees. Science Kirkman, H. and K. M. Hendrickson. 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