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

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A STUDY OF HYBRIDIZATION BETWEEN TWO SPECIES OF CYPRINID FISHES, ACROCHEILUS ALUTACEUS AND PTYCHOCHEILUS OREGONENSIS by KENNETH WILLIAM STEWART B.Sc, Colorado State University, 1958 M.Sc, Uni v e r s i t y of Miami, 1960 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Zoology We accept t h i s thesis as conforming to the required standard The University of B r i t i s h Columbia June, 1966 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced deg ree a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r ee t h a t the L i b r a r y 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 r e f e r e n c e and s t u d y . I f u r t h e r ag ree t h a t p e r m i s s i o n f o r e x -t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Depar tment o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n -c i a l g a i n s h a l l no t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depar tment o f Z o o l o g y  The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, Canada Date 30 J u n e 1966 The University of B r i t i s h Columbia FACULTY OF GRADUATE STUDIES PROGRAMME OF THE . FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY of KENNETH WILL JAM STEWART B.Sc, Colorado. State University, 1958 M.Sc, University of Miami, 1960 WEDNESDAY., JUNE 29, 1966, AT 3:30 P.M.. IN ROOM 3332, BIOLOGICAL SCIENCES BUILDING COMMITTEE IN CHARGE Chairman: I. McT. Cowan I. E. Effor d H. Norden W. S. Hoar T. G. Northcote C. C. Lindsey C. P. S. Taylor External Examiner: - R. R. M i l l e r Museum of Vertebrate Zoology University of Michigan Research Supervisor: C. C. Lindsey A STUDY OF HYBRIDIZATION BETWEEN TWO SPECIES OF GYPRINID FISHES, AGROGHEILUS ALUTAGEUS AND PTYCHOCHEILUS OREGONENSIS ABSTRACT Fi s h morphologically intermediate between Acro-cheilus alutaceus and Ptychocheilus oregonensis have been c o l l e c t e d infrequently from the Columbia River system. Morphological comparisons of wild Acrocheilus, Ptychocheilus, and intermediates, with a r t i f i c i a l i n t e r -and i n t r a - species crosses indicated that w i l d i n t e r -mediates were of hybrid o r i g i n . & Observations of spawning habitat; and behavior of the parent species suggest: that: hybridization, i n most l o c a l i t i e s i s a c c i d e n t a l . In Missezula Lake an unusuallj high incidence of hybrids, pronounced s p a t i a l separation of spawning groups of the parental species and presence of stray Ptychocheilus i n the school of Acrocheilus a l l suggest that: i n t e r s p e c i f i c mating occurs. Hybrids are l a r g e l y s t e r i l e , but. hybrid males pro-duce f e r t i l e sperm infrequently. The parent, species show no evidence of gene flow, but a few intermediates are apparently backcrosses. Gene flow i s probably blocked by the r a r i t y and p a r t i a l s t e r i l i t y of hybrids and by s e l e c t i o n against backcrosses. The presence of wild backcrosses and p a r t i a l fer-t i l i t y of hybrids suggest a large amount of genetic s i m i l a r i t y between Acrocheilus and Ptychocheilus. This contention i s strengthened by s i m i l a r i t y i n chromosome morphology and the presence of dominance e f f e c t s i n the inheritance of some parental characters i n hybrids. GRADUATE STUDIES F i e l d of Study: Ichthyology / Ichthyology Advanced Ichthyology Problems i n Ichthyology Comparative Physiology J , C. Briggs N . J . Wilimovsky C. C, Lindsey W. S. Hoar Other Studies: History of Philosophy Fi s h e r i e s Law B. Savery G. F. Curtis PUBLICATIONS Stewart, K.W., 1962. Observations on the morphology and o p t i c a l properties of the adipose eyelid of f i s h e s . J . Fish Res. Bd. Canada 19; 1161-1162. Peden, A.E., and K.W. Stewart., 1964. Extension of the known range of the a t h e r i n i d Fish Atherinops a f f i n i s . Copeia 1964; 239-240 K E N N E T H W I L L I A M S T E W A R T . A S T U D Y OF H Y B R I D I Z A T I O N B E T W E E N TWO j_ S P E C I E S O F C Y P R I N I D F I S H E S , A C R O C H E I L U S A L U T A C E U S A N D P T Y C H O C H E I L U S O R E G O N E N S I S ABSTRACT S u p e r v i s o r : C . C . L i n d s e y . Fish morphologically intermediate between Acrocheilus alutaceus and Ptychocheilus oregonensis have been co l l e c t e d infrequently from the Columbia River system. Morphological comparisons of wild Acrocheilus, Ptychocheilus, and intermediates, with a r t i f i c i a l i n t e r - and i n t r a - species crosses indicated that wild intermediates were of hybrid o r i g i n . Observations of spawning habitat and behavior of the parent species suggest that hybridization i n most l o c a l i t i e s i s accidental. In Missezula Lake an unusually high incidence of hybrids, pronounced s p a t i a l separation of spawning groups of the parental species and presence of stray Ptychocheilus i n the school of Acrocheilus a l l suggest that i n t e r s p e c i f i c mating occurs. Hybrids are l a r g e l y s t e r i l e , but hybrid males produce f e r t i l e sperm infrequently. The parent species show no evidence of gene flow, but a few intermediates are apparently backcrosses. Gene flow i s probably blocked by the r a r i t y and p a r t i a l s t e r i l i t y of hybrids and by selection against backcrosses. The presence of wild backcrosses and p a r t i a l f e r t i l i t y of hybrids suggest a large amount of genetic s i m i l a r i t y between Acrocheilus and Ptychocheilus. This contention i s strengthened by s i m i l a r i t y in chromosome morphology and the presence of dominance ef f e c t s i n the 1 1 inheritance of some parental characters i n hybrids. i i i ACKNOWLEDGEMENTS I wish to thank Dr. C.C. Lindsey for h i s help and encouragement with a l l aspects of the problem, and for h i s help i n reading and correcting of the manuscript. Dr. N.J. Wilimovsky and Dr. W.S. Hoar gave valuable advice on the problem and read and corrected the manuscript. Thanks are also due to Dr. P.M. Townsley and Dr. C.V. Finnegan for t h e i r help with the c y t o l o g i c a l studies which were part of t h i s work. Dr. J.D. McPhail was h e l p f u l with a l l aspects of the c y t o l o g i c a l studies, and es p e c i a l l y for providing i n s t r u c t i o n i n a technique he developed for preparing branchial e p i t h e l i a l c e l l s for chromosome; studies. I am es p e c i a l l y indebted to Dr. H. Tsuyuki, who ran the myogen electropherograms for me. Many people assisted i n f i e l d observations and co l l e c t i o n s , but I e s p e c i a l l y wish to thank the following people. Mr. E.E. Moodie assisted i n almost a l l the f i e l d work. Mr. A l l a n G i l l also provided help and encouragement i n the f i e l d . Mr. and Mrs. G.S. Moss provided unrestricted use of t h e i r f a c i l i t i e s at Missezula Lake. F i n a l l y , my wife assisted me i n much of the f i e l d work, i n the laboratory work, and i n preparation of the manuscript. iv TABLE OF CONTENTS Abstract i Acknowledgements i i i Table of Contents i v L i s t of Tables v L i s t of Figures v i Introduction 1 Methods 7 The Study Areas 7 F i e l d Collections and Observations ... 12 Rearing Experiments 14 Morphological Studies .. 17 Electrophoretic Studies 19 Cytological Studies 20 Results .. 23 Morphology and Electrophoresis 23 Relative Abundance of Hybrids 33 F e r t i l i t y and V i a b i l i t y 40 Cytology 43 Spawning Habitat and Behavior 50 Discussion 55 Origin of the Wild Intermediates 55 Causes of Hybridization 58 Consequences of Hybridization 63 Relationship of Ptychocheilus and Acrocheilus ... 67 Summary 70 Li t e r a t u r e c i t e d 72 V LIST OF TABLES Table Page I Frequency o f pharyngeal too th counts i n w i l d and reared p a r e n t a l spec ies and h y b r i d s . 28 I I Frequency o f occurrence o f bands i n myogen electropherograms o f w i l d p a r e n t a l spec ies and h y b r i d s . . 37 I I I Species compos i t ion o f pooled ca tch from M i s s e z u l a Lake and Wolfe Lake 39 IV Frequency o f d i p l o i d chromosome numbers f o r w i l d p a r e n t a l spec ies and reared Fj_ h y b r i d s . . 46 V Chromosome arm numbers observed f o r w i l d p a r e n t a l spec ies from Wolfe Lake and the Cheha l i s R i v e r 49 v i LIST OF FIGURES F i g u r e Page 1. D i s t r i b u t i o n o f A c r o c h e i l u s a lu taceus and P t y c h o c h e i l u s oregonensis showing known h y b r i d l o c a l i t i e s 4 2 . M i s s e z u l a Lake, showing spawning l o c a l i t i e s fo r A c r o c h e i l u s and P t y c h o c h e i l u s 8 3. Wolfe Lake and Wolfe Creek 11 4 . L a t e r a l and v e n t r a l p r o f i l e s o f A c r o c h e i l u s , P t y c h o c h e i l u s and w i l d in te rmedia tes 24 5. Numbers o f d i a g o n a l s c a l e rows i n w i l d and reared A c r o c h e i l u s , P t y c h o c h e i l u s and in te rmedia tes 25 6. Diagrams o f gut c o i l i n g pa t t e rn s fo r A c r o c h e i l u s , P t y c h o c h e i l u s , w i l d i n t e r -mediates , and j u v e n i l e A c r o c h e i l u s 26 7. R e l a t i o n s h i p s o f v a r i o u s body p r o p o r t i o n s to s tandard l eng th i n w i l d A c r o c h e i l u s , P t y c h o c h e i l u s and in te rmedia tes 27 8. Myogen electropherograms o f A c r o c h e i l u s  a lu taceus . . 34 9. Myogen electropherograms o f P t y c h o c h e i l u s  oregonensis . 35 10. Myogen electropherograms o f w i l d i n t e r -mediates 36 11 . C o l c h i c i n e t r e a t e d m i t o t i c metaphase from c u l t u r e d t e s t i s o f A c r o c h e i l u s a lu taceus . . . . 44 12. C o l c h i c i n e t r e a t ed m i t o t i c metaphase from P t y c h o c h e i l u s oregonensis c u l t u r e d t e s t i s . . . 45 13. C o l c h i c i n e t r e a t ed m i t o t i c metaphases from b r a n c h i a l e p i t h e l i u m o f reared F-^  h y b r i d s . . . 48 1 INTRODUCTION Mayr de f ines a spec ies as a p o p u l a t i o n o r group o f p o p u l a t i o n s which are r e p r o d u c t i v e l y i s o l a t e d from a l l o ther p o p u l a t i o n s . T h i s i s an o b j e c t i v e bu t not e n t i r e l y unambiguous d e f i n i t i o n . In na ture , there i s a continuum between f r e e l y i n t e r b r e e d i n g p o p u l a t i o n s and those which occur together w i thou t h y b r i d i z a t i o n . Mechanisms a f f e c t i n g the occurrence and r a t e o f gene f low between p o p u l a t i o n s have r e c e i v e d a t t e n t i o n from many i n v e s t i g a t o r s , e s p e c i a l l y i n the f i e l d of bo tany . E p l i n g (1947) i n v e s t i g a t e d the evidence f o r gene f low between p l a n t spec ies i n C a l i f o r n i a . Anderson (1949 and 1953) e x t e n s i v e l y reviewed i n t r o g r e s s i v e h y b r i d i z a t i o n i n p l a n t s . Mayr (1963) summarized knowledge r ega rd ing gene f low i n animal p o p u l a t i o n s , and many subsequent authors have modi f i ed o r e l abora ted upon h i s v i e w s . B ige low (1965) re-examines the concepts o f r e p r o d u c t i v e i s o l a t i o n and gene f low i n animal s p e c i e s . G e n e r a l l y these authors conclude tha t i f pop-u l a t i o n s are c l o s e l y r e l a t e d g e n e t i c a l l y and there i s no s e l e c t i o n a g a i n s t h y b r i d s swamping w i l l r e s u l t , g i v i n g r i s e to a p o p u l a t i o n which combines the adap t ive c h a r a c t e r i s t i c s o f bo th p a r e n t a l t ypes . I n t r o g r e s s i o n occurs when one or a few genes or gene complexes i n t r o -duced i n t o the p a r e n t a l p o p u l a t i o n by h y b r i d i z a t i o n are 2 adaptively superior to the o r i g i n a l genes or complexes. At the opposite extreme, only rare, s t e r i l e hybrids may be produced, or there may be no hybridization at a l l between related populations i n zones of contact. P r i o r to mating, behavioral, temporal and s p a t i a l i s o l a t i o n of breeding populations may l i m i t or prevent h y b r i d i z a t i o n . After mating, genetic factors causing zygote i n v i a b i l i t y or hybrid s t e r i l i t y can prevent hybrids from surviving or mating. I f hybrid to hybrid matings or backcrossing do occur, many or a l l of the progeny w i l l be selected against because recombination of the parental genotypes w i l l leave them with unbalanced and poorly adapted genotypes. Compared with other animals, f i s h hybridize rather frequently. Hubbs (1955) points out that hybrids are more common in freshwater than i n marine fishes, in temperate and boreal areas than in the tropics, and i n depauperate than i n speciose f i s h faunas. The North American cyprinids are among the more thoroughly studied groups of fishes with regard to natural h y b r i d i z a t i o n . Hubbs, alone and with several co-workers has done much of t h i s work. G i l b e r t (1961) investigated the r e l a t i o n s h i p of Notropis cornutus and Notropis  chrysocephalus chrysocephalus i n several zones of contact of the two species of Michigan. In large part, the e x i s t i n g studies of cypr i n i d h y b r i d i z a t i o n have been confined 3 to analysis of f i e l d c o l l e c t i o n s of the parents and intermediates. A r t i f i c i a l l y produced F i hybrids i n the etheostomatine fishes have been reared i n the laboratory by Hubbs (1957, 1958 and 1959), Hubbs and Hubbs (1932a, 1932b and 1933) and have reared Fj_ a r t i f i c i a l crosses of Lepomis species and Mollienesia l a t i p i n n a x M. mexicana. Hybridization among the P a c i f i c Northwestern cyprinid genera was f i r s t reported by Hubbs and Schultz (1931) , for Acrocheilus alutaceus. x Ptychocheilus oregonensis. Weisel (1954, 1955a and 1955b) reported combinations involving Richardsonius, Ptychocheilus, Mylocheilus and Rhinichthys. A l l of these studies were based s o l e l y upon morphological! analysis of wild specimens. The|present study deals with h y b r i d i z a t i o n between species of two nominal genera of cyprinid fishes, the northern squawfish, Ptychocheilus oregonensis (Richardson) and the chiselmouth, Acrocheilus alutaceus (Agassiz and Pickering). Ptychocheilus oregonensis and Acrocheilus alutaceus both are abundant i n the Columbia River system. Ptychocheilus also occurs throughout the Fraser and Skeena River systems, and in a l l but a few coastal streams of Washington and B r i t i s h Columbia. Acrocheilus, however, i s scattered i n occurrence i n B r i t i s h Columbia, with the exception of the Okanagan River (Fig. 1) . Intermediates between these two species have been taken infrequently since 1905 i n the Columbia River system in the United States. Snyder (1905 4 F i g „ 1, D i s t r i b u t i o n o f A c r o c h e i l u s a lu taceus and P t y c h o c h e i l u s oregonensis showing known h y b r i d l o c a l i t i e s . M 100 Km. H 100 miles A Hybr id L o c a l i t i e s • D i s cont inuous A c r o c h e i l u s Loca l i t ie s — Bounda ry of Cont inuous A c r o che i l u s D i s t r ibut ion * - * Boundary of P t ychoche i l u s o regonens i s D istr ibut ion 5 and 1908) recorded an intermediate as being equivalent to the Leuciscus caurinus of Richardson (1836). Hubbs and Schultz (1931) on the basis of Snyder's specimen and some new material attributed t h i s form to h y b r i d i z a t i o n between Acrocheilus and Ptychocheilus. Schultz and Schaeffer (1936) and Schultz and De Lacey (1936) referred to a combination between Acrocheilus and Mylocheilus, c i t i n g Hubbs and Schultz (1931). This, i n fact, i s the Acrocheilus x Ptychocheilus combination. No subsequent references to t h i s form appeared u n t i l Patten (1960) reported i t s occurrence i n the Yakima River, Washington. The present study has attempted f i r s t to v e r i f y that the wild intermediates are the r e s u l t of h y b r i d i z a t i o n . To t h i s end, morphological comparisons were made for wild and reared f i s h of both parental species and the hybrids. The probable causes of h y b r i d i z a t i o n were studied by means of f i e l d observations of the spawning habitat and behavior of the parental species. Also, gamete l i f e was studied i n order to determine the p o s s i b i l i t y of h y b r i d i z a t i o n due to chance meeting of d r i f t i n g gametes. F i n a l l y , the p o s s i b i l -i t y of gene flow between the parental species was investigated. The parental species and hybrids were compared for a number of morphological characters, and for muscle e l e c t r o -pherograms. Chromosome number and morphology i n the parents and hybrids were studied, and hybrid f e r t i l i t y and the existence of n a t u r a l l y occurring backcrosses were 6 investigated. Comparisons of the r e l a t i v e abundances of hybrids to parental species were made in order to determine the p o s s i b i l i t y of a s i g n i f i c a n t amount of gamete wastage due to hyb r i d i z a t i o n . 7 METHODS A. The Study Areas Missezula and Wolfe Lakes and their inlet and outlet streams were the primary study areas. Both of these lakes drain into the Columbia River system via the Similkameen and Okanogan Rivers. The remaining l o c a l i t i e s , from which samples of juvenile and adult fish were collected, are the following. 1. Vidette Lake, which drains into the Fraser River system via the Thompson River. 2. Lome and Izzits Lakes, which are respectively the uppermost and middle lakes in the Wolfe Creek drainage which also includes Wolfe Lake. 3. The Okanogan River, between the towns of Riverside and Brewster, Washington.1 Missezula Lake is located 25 miles by road north of Princeton, B r i t i s h Columbia. It drains into the Similkameen River via Summers Creek and Allison Creek. Missezula is the larger of the two intensively studied lakes, being 7.3 Km. long, a maximum of 600 M. wide and a maximum depth of 65.3 M. (Fig. 2). There are several streams draining into Missezula Lake, only one of which, Leonard Creek, flows constantly. Leonard Creek is partly obstructed by beaver 8 F i g . 2. Missezula Lake, showing spawning l o c a l i t i e s for Acrocheilus and Ptychocheilus. 9 dams near i t s mouth and i s quite cold. Its temperature was recorded i n l a t e June at 9 .4°C. No cyprinids were ever seen spawning i n the flowing part of Leonard Creek, although Richardsonius balteatus was observed i n spawning condition in the shallow, weedy area of the lake around i t s mouth. Since none of the other creeks were flowing during the time in which cyprinids were i n spawning condition (late May to early July) Leonard Creek o f f e r s the only opportunity for i n l e t spawning. The o u t l e t of Missezula Lake i s blocked by a low earth dam which raises the l e v e l of the lake by a minimum of 1.2 M. The dam has also had the e f f e c t of a l t e r i n g the narrow area immediately above i t from a shallow gradient stream bed to an area of standing water up to 1.8 M. i n depth. Rounded stream worn stones could be found i n the center of t h i s area beneath approximately 60 cm. of accumu-lated s i l t . There i s a d i r e c t i o n a l current along the shores of the o u t l e t area which attains up to 15 cm. per second v e l o c i t y . The bottom near the shore consists of angular gravel which i s kept free of s i l t , probably by the current. I t i s i n t h i s area that Acrocheilus and intermediates i n spawning condition were taken. The dam does not con-s t i t u t e a b a r r i e r to f i s h movement, either into or out of the lake. Eastern brook trout were observed passing i n both d i r e c t i o n s i n overflow channels around the spillway. No cyprinids were either c o l l e c t e d or observed i n the stream 10 below the dam during the study however, so i t may be assumed that a l l spawning f i s h are confined to the lake proper or the region immediately above the dam (Fig. 2). Wolfe Lake i s 10 miles by road east of Princeton. I t l i e s near the mouth of a v a l l e y opening into the Similkameen canyon, and drains into the Similkimeen River v i a a segment of Wolfe Creek about 1.6 Km. long. Wolfe Lake i s roughly circular,: about 600 M. i n diameter, and the depth probably does not exceed 6.1 M. (Fig. 3). Wolfe Lake has a single i n l e t stream, Wolfe Creek, which continues below the lake as the o u t l e t . No other surface drainage either enters or leaves the lake. The i n l e t reached a temperature of 19.4°C. i n l a t e June, only 1 degree cooler than the lake surface temperature and the out l e t temperature. Around the mouth of the i n l e t there i s an extensive marsh and the stream gives o f f a number of small channels, forming a del t a . Above the,marsh area the stream gradient i s r e l a t i v e l y shallow with pools separated by r i f f l e sections. The banks of the i n l e t stream are covered with a heavy growth of brush over i t s entire length, and there are frequently p i l e s of brush sunken i n pools. Mr. E.E. Moodie observed a sharp decline i n catch of adult cyprinids i n the lake at the time spawning f i s h appeared i n the stream, so i t may be assumed that most of the cyprinids i n the lake u t i l i z e the stream for spawning. 11 F i g . 3 . Wol fe Lake and Wol fe C r e e k . Wolfe Lake and Wol fe C r e e k Scale: ,, 3 0 0 meters, • - Col lect ion &. Observa t ion Station x - A c r o c h e i l u s & Ptychochei lus Upper Limit in C reek 12 The out l e t of Wolfe Lake, l i k e Missezula Lake, has been blocked by an earth dam. The drop between,the lake l e v e l and the l e v e l of the stream below i s greater i n Wolfe Lake, and, while the dam may not be a b a r r i e r to the move-ment of adult f i s h , i t i s probably an e f f e c t i v e b a r r i e r to upstream movement of young cyprinids. In addition, the gradient of the outl e t stream i s steeper than that of the i n l e t , making i t less probable that young cyprinids would be able to return to the lake. B. F i e l d Collections and Observations Samples of adult f i s h were obtained by g i l l n e t t i n g , both from spawning and non-spawning f i s h . Seining and rotenone, as well as small-meshed g i l l n e t s were used i n c o l l e c t i n g juvenile specimens. When sampling standing water, stationary g i l l n e t sets were used, l e f t i n most cases for a period of ten hours (overnight). In flowing water, the g i l l n e t s were allowed to d r i f t through the desired area and the f i s h removed immediately afterward. Seining was of r e s t r i c t e d use i n most areas studied due to the presence of logs and boulders on the bottom. The most e f f e c t i v e c o l l e c t i o n s of juvenile f i s h were obtained either with rotenone or fine-mesh g i l l n e t s . G i l l n e t c o l l e c t i o n s were made d a i l y during both the 1964 and 1965 f i e l d seasons from Missezula Lake. A l l 13 hybrids captured were saved for morphological analysis. Most of the Acrocheilus and Ptychocheilus used i n the morphological studies were taken from Missezula Lake i n 1964, although c o l l e c t i o n s from a l l areas studied were compared with these. Fish which were retained a l i v e for l a t e r work were kept i n stryofoam cooler chests for transport to the shore or laboratory, and f i s h which were to be retained a l i v e for longer than a few days were treated with an intraperitoneal i n j e c t i o n of 1,000 units of p e n i c i l l i n and 1,000 micrograms streptomycin (Difco Penicillin-Streptomycin T.C.) i n order to prevent i n f e c t i o n i n net wounds. Fish to be preserved had t h e i r body c a v i t i e s opened on the r i g h t side and were preserved i n 10% Formalin immediately a f t e r c o l l e c t i o n . Samples of gonads for h i s t o l o g i c a l studies were preserved i n Carnoy's f i x a t i v e (3 parts ethanol :1 part g l a c i a l a c e t i c acid) and r e f r i g e r a t e d . Groups of f i s h i n spawning condition were observed both from above water and underwater. In Missezula Lake a small p i e r was b u i l t i n the o u t l e t adjacent to the area where f i s h were spawning. Observations were also made suc c e s s f u l l y by d r i f t i n g along the shore of the lake i n a boat, the water being cl e a r enough for good v i s i o n to a depth of 4.6 - 6.1 M. Underwater observations were confined to Wolfe Creek and the o u t l e t of Missezula Lake. In the nearly stationary water of the lake, i t was necessary to 14 use an oxygen rebreather, since the noise produced by open c i r c u i t d i v i n g equipment or even by breathing through a snorkel tube frightened the f i s h . In Wolfe Creek, the flowing water produced s u f f i c i e n t background noise so that the f i s h were not disturbed by the breathing noises caused by a snorkel tube. Data from underwater observations were either recorded i n p e n c i l on a sheet of p o l y v i n y l p l a s t i c while underwater or recorded with other f i e l d notes a f t e r the dive. C. Rearing Experiments Wild adult f i s h of both parental species as well as hybrids, when available, were used as parents for experimentally reared young. A l l but two of the parent f i s h were obtained from Missezula Lake. A t o t a l of 44 a r t i f i c i a l crosses were made during the 1964 and 1965 seasons. The types of crosses and t h e i r fate are as follows. During the 1964 season, 33 a r t i f i c i a l crosses were made. Of these, 16 crosses hatched s u c c e s s f u l l y and yielded young which could be used for morphological studies. They comprised 8 Acrocheilus x Acrocheilus crosses, 10 female Acrocheilus x male Ptychocheilus reciprocals and one backcross of female Acrocheilus x male presumed Fi_ x Ptychocheilus parentage. Two female Ptychocheilus x 15 male Acrocheilus reciprocals died after 72 hours of development at 20°C, as did 6 Acrocheilus x Acrocheilus and 6 female Acrocheilus x male Ptychocheilus crosses. During the 1965 season, a total of 11 crosses were made. Three of these produced young which were used for morphological analysis. These were 2 female Ptychocheilus x male Acrocheilus hybrid reciprocals and 1 female Acrocheilus x male Ptychocheilus reciprocal . In addition, young from 2 Ptychocheilus x Ptychocheilus crosses made by C.C. Lindsey were used. One of these originated from Lac la Hache parents, which are al lopatric from Acrocheilus. Three wild presumed F]_ hybrids with free gametes were taken in 1965. Two of these were males, and each was crossed with two Acrocheilus females. None of the eggs survived beyond 72 hours. A single female presumed F i with free eggs was crossed with both a male Acrocheilus and a male Ptychocheilus. A l l but 200 of the resulting eggs were dead within 60 minutes of f e r t i l i za t i on , and the remaining 200 a l l died after 24 hours of development at 18°C. A single cross of a female Richardsonius balteatus x male Acrocheilus yielded one offspring whose morphology was studied. Parent f ish were taken in overnight g i l lnet sets near areas were aggregations of fish in spawning condition or actual spawning had been observed. The f ish were retained l ive in styrofoam cooler chests and brought to 16 shore. The eggs of a given female were stripped onto nylon c r i n o l i n e screen i n a dry stacking dish i n l o t s of from one to three hundred per dish. In a l l cases, eggs from a single female were f e r t i l i z e d with sperm from at l e a s t one eonspecifie and one hete r o s p e c i f i c male. Also a given male, where possible, was crossed with a female of both species. After f e r t i l i z a t i o n , the eggs of each l o t were spread into a single layer on the c r i n o l i n e screen which was then c a r e f u l l y immersed i n water i n a second stacking dish. The eggs were always shielded from d i r e c t sunlight. During the spawning season| i n 1964, the eggs were transported by a i r to Vancouver to be hatched i n the laboratory. In 1965, most of the eggs were kept i n screen enclosed boxes i n the lake for hatching. The eggs were held i n 7 X 9 x 10 em. baskets made of nylon c r i n o l i n e and l i n e d with nylon c h i f f o n . These were suspended i n the water by means of a wooden frame inside the rearing box. A f t e r the eggs hatched, the young f i s h were retained i n the c r i n o l i n e baskets for about two months during the 1964 season, or transferred to 20 l i t e r aquaria two weeks af t e r hatching during the 1965 season. Much of the pre-hatching mortality was apparently due to i n s u f f i c i e n t oxygen i n the water i n the c r i n o l i n e baskets. Since fine mesh l i n e r s are required i n order to r e t a i n the newly hatched f i s h , heavy aeration i n the rearing box or aeration of each c r i n o l i n e basket i s necessary. The c i r c u l a t i o n within the screen enclosed rearing boxes held in 1 7 M i s s e z u l a Lake d u r i n g the 1965 season was not s u f f i c i e n t to a v o i d m o r t a l i t y . In f a c t , the m o r t a l i t y to eggs h e l d i n these boxes was h e a v i e r than t h a t o f eggs hatched i n the l a b o r a t o r y . A f t e r h a t c h i n g , the young f i s h r e q u i r e d about 5 days to absorb t h e i r remain ing y o l k (at 1 8 - 2 0 ° C . ) . They were then fed a mix tu re o f b r i n e shrimp (Artemia) n a u p l i i and i n f u s o r i a c u l t u r e i n the case o f those rea red i n the Vancouver l a b o r a t o r y , or the i n f u s o r i a c u l t u r e and n a t u r a l l y o c c u r r i n g p l a n k t o n ( l a r g e l y Diaptomus) fo r f i s h reared i n the f i e l d . As the f i s h grew, the d i e t was supplemented w i t h d r y t r o p i c a l f i s h food ( "Te t ra -Min Growth Food") i n i n c r e a s i n g p r o p o r t i o n s , and the f i s h were main ta ined e n t i r e l y on t h i s food a f t e r they had reached 2-2% months o f age. A l l e x p e r i m e n t a l l y reared f i s h were kep t a l i v e u n t i l the f i n s were f u l l y formed, and when p o s s i b l e , u n t i l the s c a l e s were formed. D. M o r p h o l o g i c a l S tud ies Both w i l d and e x p e r i m e n t a l l y rea red f i s h were used fo r morpho log i ca l s t u d i e s . F i e l d i d e n t i f i c a t i o n o f the paren t spec ies and h y b r i d s i s not d i f f i c u l t and notes r ega rd ing c o l o r , presence o f spawning t u b u r c l e s and genera l appearance c o u l d be made r e a d i l y on uncaptured f i s h e i t h e r w h i l e d i v i n g o r obse rv ing from the s u r f a c e . In the case o f s m a l l f i s h (20 mm. or smal le r ) i t 18 was necessary to s t a i n the specimens with a l i z a r i n e red-s dye i n order to count the scales and pharyngeal teeth. Measurements on these small f i s h were made using a stereomicroscope equipped with a graduated mechanical stage and a crosshair i n the ocular. Larger f i s h were measured with d i a l c a l i p e r s to the nearest 0.1 mm. Sample sizes of f i s h for each morphological character are indicated on the figure or table r e l a t i n g to that character. A t o t a l of 2,622 adult cyprinids were co l l e c t e d during the 1964 and 1965 seasons. Only a portion of these were preserved and returned to Vancouver. Most characters are defined i n accordance with Hubbs and Lagler (1947). The exceptions are given below. 1. La t e r a l scale rows are the number of diagonal scale rows crossing the l a t e r a l l i n e . The anteriormost scale row counted had the l a t e r a l l i n e scale i n contact with the pectoral g i r l d e . The posteriormost row counted terminated with l a s t l a t e r a l l i n e scale on the base of the caudal f i n . This count was found to be easier than was a count of pored l a t e r a l - l i n e scales on Acrocheilus, on which the scales are imbedded, and on very small f i s h . 2. Gut length measurements are not d e t a i l e d 19 i n Hubbs and L a g l e r (1947). The gut was removed by s e v e r i n g a t the t r ansve r se septum a n t e r i o r l y and c u t t i n g the body w a l l around the anus p o s t e r i o r l y . A r b i t r a r i l y , the stomach was de f ined as the a n t e r i o r l o n g i t u d i n a l segment, and a l l p o r t i o n s from the f i r s t cu rva tu re to the anus were des igna ted as i n t e s t i n e . E . E l e c t r o p h o r e t i c S tud ies E l e c t r o p h o r e t i c a n a l y s i s o f muscle p r o t e i n s was c a r r i e d out by D r . Tsuyuk i o f the F i s h e r i e s Research Board o f Canada T e c h n o l o g i c a l S t a t i o n i n Vancouver . W i l d f i s h o f both paren t spec ies and the h y b r i d s were c o l l e c t e d i n the f i e l d fo r t h i s purpose . The f i s h were r e t a i n e d a l i v e u n t i l the sample was taken . The sample c o n s i s t e d o f a f i l l e t cu t from the l o n g i t u d i n a l muscle mass which was l a b e l e d and f rozen on CO2 i c e immediate ly a f t e r b e i n g removed from the f i s h . The samples remained f rozen u n t i l a n a l y s i s , a t which t ime the s k i n and dark muscle was removed and d i s c a r d e d . The remain ing wh i t e muscle was broken up i n water by means o f a b l ende r , and the c l e a r f i l t r a t e from t h i s mix tu re was used to make the e l e c t r o p h o r e t i c r u n . A f t e r deve lop ing , the r e s u l t i n g pa t t e rn s were photographed and the photographs used i n 20 making comparisons. F. Cytological Studies Chromosome number and morphology were determined for both parent species and for experimentally reared hybrids representing both r e c i p r o c a l crosses. Two techniques were used successfully to obtain chromosome preparations. The f i r s t of these was an adaptation of f i s h tissue c u l t u r i n g techniques as reported by Parker (1950), Wolfe and Dunbar (1956), Wolf e et a l (I960), Townsley et a l (1963) and Roberts (1964). Primary cultures of t e s t i s were obtained from both parent species by asepti-c a l l y removing the testes, mincing them into pieces 1 mm. or less i n diameter i n s t e r i l e f i s h Ringer's solution, and explanting from f i v e to f i f t e e n pieces into a 30 ml. culture f l a s k (Falcon P l a s t i c s s t e r i l e , disposable 30 ml tissue culture f l a s k ) , 2.ml. of B.B.L. tissue culture medium 199, Hank's base, plus 10% human serum was added to the fl a s k and the culture incubated at 18°C for seven days. After the incubation period, 2 ml of fresh medium and 0.01 ml. of 0.1% co l c h i c i n e were added to the culture. After an additi o n a l incubation period of 36 hours, the tissues were treated for 10 minutes with a hypotonic solution consisting of 1 part medium to 6 parts d i s t i l l e d water. F i x i n g and staining were done simultaneously i n 2% l a c t i c - a c e t i c 21 orcein(Natural orceins2 gm, Acetic acid: 45 ml, l a t i c acid 55 ml). Smears were prepared immediately by scraping a suspension of c e l l s from the piece of tissue into a drop of s t a i n on a cleaned s l i d e . A coverslip (22 mm.square #0) was placed over the drop of stain, a i r bubbles excluded by l i g h t pressure with a di s s e c t i n g needle, and the preparation squashed by the application of the author's f u l l weight on a #4 rubber stopper placed on the coverslip. Examination and photography were done using a Zeiss photomicroscope equipped for phase contrast. A l l photos were made using the o i l immersion objective. Due to the extremely small s i z e of the gonad in the reared hybrids, i t was not possible to s t a r t a culture. For these specimens, a technique developed by J.D. McPhail (per. comm.) was employed. This involved the use of e p i t h e l i a l c e l l s from the lamellae of the fourth g i l l arch. The f i s h (about 2 cm.in length) were treated with a subcutaneous i n j e c t i o n of 0.05 ml of 0.01% colchi c i n e i n f i s h Ringer's and held a l i v e i n heavily aerated water for 1 hour. The fourth g i l l arch was then removed i n t a c t and placed i n d i s t i l l e d water for 30 minutes and i n l a c t i c -a c e t ic orcein for an additional 15 minutes. A c e l l suspension was then scraped from the terminal portions of the lamellae into a drop of s t a i n on a s l i d e and the smear made as described previously. Chromosome morphology was compared between the 22 parent species and hybrids by means of photographs. A karyotype for each parent species, showing the probable p a i r i n g of the chromosomes, was constructed by arranging the chromosomes according to total': length, centromere posi t i o n , and the presence of co n s t r i c t i o n s . Comparisons were then made between the parent species, and hybrids. D i p l o i d number and arm number could be determined with more cert a i n t y than the morphology of s p e c i f i c chromosomes with the r e s t r i c t e d amount of material which was ava i l a b l e . Some of the chromosomes were d i s t i n c t i v e because of t h e i r s i z e or morphology; however,.and these could be seen i n a l l the preparations. 23 RESULTS A. Morphology and Electrophoretic Studies The morphological differences between Acrocheilus and Ptychocheilus may be summarized as fpllows:-1. Scale counts: higher i n Acrocheilus than Ptychocheilus, with l i t t l e overlap (Fig. 4). 2. Pharyngeal teeth: Acrocheilus has a single row of teeth on each pharyngeal arch with 4 or 5 teeth (0,4-5,0). Ptychocheilus has 2 rows of teeth per arch, the most common count being 2,5-4,2, with rare variants showing 1,5-4,2; 2,5-4,3 or 3,5-4,2. (Table I ) . 3. I n t e s t i n a l t r a c t : complexly c o i l e d i n Acrocheilus (Fig. 5), a simple S-shaped tube in Ptychocheilus (Fig. 5). 4. Peritoneum color: black i n Acrocheilus, s i l v e r y i n Ptychocheilus. 5. Lower jaw: has a keratinized, c h i s e l - l i k e anterior margin i n Acrocheilus, and i s rounded, with no s p e c i a l i z a t i o n i n Ptychocheilus (Fig. 6) . 6. Body proportions: Acrocheilus has a narrower caudal peduncle, shorter jaws and shorter snout than Ptychocheilus (Fig. 7). 24 F i g . 4 0 L a t e r a l and ventral p r o f i l e s of Acrocheilus, Ptychocheilus and wild intermediates. 1. Ptychocheilus oregonensis, Missezula Lake. 2 0 Presumed F]_ x Ptychocheilus, Missezula Lake. 3. F]_ Hybrid, Missezula Lake. 4. Presumed F]_ x Acrocheilus, Wolfe Lake. 5„ Acrocheilus alutaceus, Missezula Lake. 25 F i g . 5. Numbers of diagonal scale rows i n wild and reared Acrocheilus, Ptychocheilus and Intermediates. Horizontal l i n e = range; v e r t i c a l l i n e = mean; s o l i d rectangle = 2 standard deviations. Wild Ac roche i lus Rea red Ac roche i lus Wild P r e sumed_ RjX Ac roche i l us Wild R, Hybr ids R e a r e d F. Hybr ids Wild P resumed F 1 X Ptychoche i lus Wild Sympatr ic P tychoche i lus Wild A l lopatr ic P tychoche i lus 6 0 .N=48 .N=3 1 N=2 .N=40 .N=23 1 N=2 ,N=49 ,N=20 — I — 9 0 7 0 8 0 Number of Diagonal Sca le Rows (Solid Rectang les = 2 Standard Deviations) 100 26 F i g . 6. Diagrams of gut c o i l i n g patterns for Acrocheilus, Ptychocheilus, wild intermediates, and juvenile Acrocheilus. 27 F i g . 7. R e l a t i o n s h i p s o f v a r i o u s body p r o p o r t i o n s to s tandard l e n g t h i n w i l d A c r o c h e i l u s , P t y c h o c h e i l u s and In te rmed ia tes . o Rj Hybrids • Ac roche i l u s A Sympat r i c Ptychochei lus A A l l opa t r i c Ptychochei lus • F X Ptychochei lus a fij X Acrocheilus • • •• •• ^ A A ^ A A ^ O * 4 A * ° > T ^ A <A 2 °° ° 5 0 100 150 Standard Length mm. 2 0 0 2 5 0 o o c c c <u a _i *-* .10-c °b .05^  • A * * A*A A  A A *A4 AA QOOS O O ° o . 8° tr °o ° °o .0 O O op o o o o J 5 0 100 150 Standard Length mm. 2 0 0 2 5 0 . 5 0 i 3 C O fl) .25 A A A A O i . » A . A A , A O , A ^ f AA O oooo <5>o a> oo 5 0 100 150 Standard Length mm. 2 0 0 2 5 0 T a b l e I F r e q u e n c y o f p h a r y n g e a l t o o t h counts i n w i l d and r e a r e d p a r e n t a l s p e c i e s and h y b r i d Acrocheilus Fl H Y B R I D S B A C K C R O S S E S Ptychocheilus Number of outer row pharyngeal teeth L e f t Right Arch Arch Wild Reared Female Acrocheilus Wild Female Ptychocheilus Wolfe Lake Presumed F l * Acrocheilus Missezula Lake Presumed F X x Ptychocheilus Presumed male F^ x Ptychocheilus Sympatric Wild Reared A l l o p a t r i c Wild x male Ptychocheilus x male Acrocheilus X female Acrocheilus reared Reared Wild Wild Reared 0 - 0 56 18 40 0 - 1 1 3 1 - 0 1 2 1 - 1 14 4 1 2 15 1 - 2 2 5 1 2 - 1 4 7 1 1 2 - 2 6 26 3 1 2 59 35 1 2 - 3 2 18 3 - 2 2 1 1 3 - 3 1 Sample sizes 56 18 28 42 4 2 2 63 63 37 20 29 The body p r o p o r t i o n s o f the paren t spec ies are s i m i l a r i n s m a l l f i s h and d i v e r g e w i t h i n c r e a s i n g s i z e . The o n l y r e l i a b l e cha rac te r s f o r d i s t i n g u i s h i n g paren t spec ies and in te rmedia tes below one year i n age are the pharyngeal too th counts , the s c a l e counts i f s ca l e s are formed, and the p e r i t o n a l c o l o r . The c h i s e l l i p i s not apparent i n A c r o c h e i l u s u n t i l the end o f the f i r s t summer a t the e a r l i e s t , and i s rounded i n o u t l i n e i n f i s h below 4-6 cm. Before the c h i s e l has appeared, A c r o c h e i l u s tends to have a s l i g h t l y sub te rmina l mouth, which a id s i n d i s t i n -g u i s h i n g i t from the squawfish, bu t t h i s may be masked by deformat ion i n p rese rved f i s h . The in t e rmed ia t e forms c o l l e c t e d i n the w i l d f f a l l i n t o th ree c a t e g o r i e s . By f a r the most common form agrees comple te ly w i t h the d e s c r i p t i o n s by Snyder (1906), Hubbs and S c h u l t z (1931) and Pa t t en (1960). The f i g u r e s and t a b l e s r e f e r r e d to i n the d i s c u s s i o n o f pa ren t spec ies morphology g i v e the va lues o f a l l three h y b r i d morphologies fo r each c h a r a c t e r . One o f the s t r i k i n g fea tures o f the morphology o f the common in t e rmed ia t e type i s the apparent dominance o f some squawfish c h a r a c t e r s . No t r a c e o f a k e r a t i n i z e d edge i s to be found on the lower jaw, a l though i n most o ther r e spec t s the mouth p r o f i l e i s in t e rmed ia te ( F i g . 6 ) . The i n t e s t i n e shows none o f the c o i l i n g to be seen i n A c r o c h e i l u s , a l though the a n t e r i o r loop o f the i n t e s t i n e 30 i s longer than i n Ptychocheilus. In some cases, i n f l e c t i o n s are present a n t e r i o r l y which are reminiscent of the gut morphology i n young-of-the-year Acrocheilus. The pharyngeal teeth of wild intermediates are most commonly i d e n t i c a l i n count to Ptychocheilus, although a few individuals show a 1,5-4,1 count, which might a r b i t r a r i l y be considered intermediate between 0,5-4,0 and 2,5-4,2. Most other characters l i e between mean values of the parent species. The peritoneum, (data not shown) varies from s i l v e r y with d i f f u s e melanophores to almost black, with a s i l v e r y sheen. Most commonly, i t i s grey. The other two intermediate types c o l l e c t e d were represented by four specimens, two taken from Missezula Lake, and two from Wolfe Lake. The Missezula Lake specimens d i f f e r e d from t y p i c a l intermediates i n having lower scale counts, 73 and 74 diagonal rows, a much shorter anterior loop of the gut, and a s t r a i g h t rather than decurved mouth (Fig. 6). They tended therefore, to be more Ptychocheilus-l i k e . The Wolfe Lake specimens were, on the other hand, more Acrocheilus -like. The> scale counts were 83 and 85, the gut was strongly i n f l e c t e d , (Fig. 5), but not c o i l e d , and the lower jaw had a rounded, keratinized margin (Fig. 6 ) . The mouth was sharply deflected, as i n Acrocheilus, rather than decurved, as i n the t y p i c a l intermediates. The pattern observed i n nature, then, consists of the two parent forms, and three r e a d i l y distinguishable 31 intermediate forms, two of which are extremely rare. Since the body proportions are not d i s t i n c t i v e i n youhg-of-the-year f i s h , analysis of the morphology of reared specimens was l i m i t e d for the most part to pharyngeal tooth counts and scale counts, both of which are d i s t i n c t i v e i n the natural i populations. Reared Acrocheilus a l l showed 0,5-4,0 pharyngeal tooth count, as do the wild f i s h (Table I ) . The mean scale count of the only three reared Acrocheilus which survived long enough for scales to be formed was 85.66 (range 83-86) as compared to a mean of 85.83, with a range of 80-94 for wild Acrocheilus (Fig. 4). In the three f i s h i n which scales had:formed (about 3 cm. body length) a rounded, keratini z e d margin was beginning to appear on the lower jaw. This was c l o s e l y comparable to that observed i n wild young-of-the-year Acrocheilus i n Missezula Lake during the same month (October). The gut showed i n f l e c t i o n s much l i k e those diagrammed for 38 mm. wild f i s h (Fig. 5). The reared squawfish originated from Wolfe Lake and Lac l a Hache parents, and were reared at three d i f f e r e n t constant temperatures. None was reared to a large enough si z e for scales to have formed, but pharyngeal tooth counts were r e a d i l y made on a l l (Table I ) . The counts are the same as those observed i n wild Wolfe Lake and Missezula Lake populations. The Lac l a Hache specimens (shown separately i n Table I ) are a l l o p a t r i c , so that there i s no p o s s i b i l i t y 32 of introgression of chiselmouth genes Into t h i s population. Table I shows the pharyngeal tooth counts of reared F^ hybrids i n comparison with wild intermediates and the parent species. The two re c i p r o c a l crosses are shown separately. The data for the female Acrocheilus r e c i p r o c a l represent the pooled r e s u l t s of a l l crosses of t h i s type which were made. Only f i v e o f f s p r i n g of the female Ptychocheilus re c i p r o c a l , representing a single cross, survived. There i s a difference i n the modal pharyngeal tooth count of reared F^s of the female Acrocheilus r e c i p r o c a l and the wild intermediates. The former are predominantly 1-1 and the l a t t e r 2-2. This may,be due i n part, to the small s i z e of many of the reared F^s, which died at about 1.5 cm., before scales were formed. The f i v e female Ptychocheilus reciprocals which were hatched successfully were reared to about 3 cm., and, of those, three showed a 2-2 count as i s seen i n the wild intermediates. Scale counts of 18 female Acrocheilus reciprocals had a mean of 79.77, compared to a mean of 80.58 for the wild intermediates. This difference i s not s i g n i f i c a n t at the 95% confidence l e v e l (Fig. 4). The f i v e female Ptychocheilus reciprocals had a mean scale count of 80.4, almost i d e n t i c a l to the wild intermediates. Reared specimens, then correspond c l o s e l y i n t h e i r morphology to wild specimens presumed to be of the same parentage. 3 3 In order to compare the muscle electropherograms of the parental species and hybrids, the bands present were designated with l e t t e r s , beginning with A at the cathode side of the gel. The frequency of occurrence of each band present was then determined for the parental species and hybrids (Table I I ) . Photographs of the electropherograms of a l l usable samples are given i n Figs. 8, 9 and 10. A number of the samples which originated from Lome and Vidette Lakes and the Okanogan River were held i n frozen storage f o r about three months before being run. The patterns obtained from these samples were anomalous, indi c a t i n g that decomposition had occurred during storage. No consistent differences could be found between Acrocheilus and Ptychocheilus, electropherograms. (Table II; Figs. 8, 9 and 1Q). B. Relative Abundance of Hybrids The quantitative composition of the catch by species was recorded for a l l samples of adult f i s h from each study area. Samples of juveniles were also analyzed for species composition, but are more r e s t r i c t e d i n the numbers of f i s h c o l l e c t e d and i n the area sampled. Also, i t was mentioned previously that i d e n t i f i c a t i o n of hybrids i s uncertain i n young-of-the-year f i s h , so that estimates of hybrid abundance i n juveniles i s probably not as r e l i a b l e 34 Fig„ 8. Myogen electropherograms of Acrocheilus alutaceus. Acrocheilus alutaceus Missezula Lake 15cm. • Wolfe Lake Okanogan River Vidette Lake 35 F i g . 9 . Myocren electropherograms of Ptychocheilus  oregonensis. Ptychocheilus oregonensis Vidette Lake 3 6 F i g . 10. Myogen electropherograms of w i l d intermediates. Wild Intermediates Presumed Ej X Acroche i lus Wolfe Lake Table II Frequency of occurrence of bands in myogen electropherograms of wild parental species and hybrids. L E F T R I G H T Missezula Lake Acrocheilus  Ptychocheilus Fj_ Hybrids Wolfe Lake Acrocheilus  Ptychocheilus Fj_ Hybrids F]_ x Acrocheilus Lorne Lake Ptychocheilus Vidette Lake Acrocheilus  Ptychocheilus Okanogan River Acrocheilus Fraser River Ptychocheilus Pooled Results Acrocheilus Ptychocheilus 13 9 13 9 12 9 3 13 13 1 11 to F]_ Hybrids Fj_ x Acrocheilus A B C D E F G o r i g i n H I J K 2 2 2 2 2 2 2 2 2 8 6 8 5 8 8 8 8 8 1 1 1 1 1 1 1 2 1 . 1 2 1 2 2 2 none sampled — — ' • - - - - - - - - -1 1 1 1 1 1 1 1 . 1 1 1 1 . 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 . 1 1 1 1 1 . 1 1 8 6 8 6 8 7 6 8 8 6 2 . 1 2 2 2 2 2 1 1 1 1 1 1 1 38 as are the estimates for adult f i s h . Missezula Lake had the highest r e l a t i v e abundance for adult hybrids, 14.58 per cent r e l a t i v e to Acrocheilus. In Wolfe Lake, hybrids constituted 1.31 per cent r e l a t i v e to the Acrocheilus. The samples on which these figures are based include a t o t a l of 110 Acrocheilus and hybrids c o l l e c t e d i n d a i l y samples over a three month period i n Missezula Lake and 232 Acrocheilus and hybrids c o l l e c t e d in d a i l y samples over a one month period i n Wolfe Lake (Table I I I ) . A s i n g l e sample of juveniles from the Okanogan River, near Brewster, Washington contained 13.2 percent hybrids r e l a t i v e to Acrocheilus juveniles. No adult hybrids were taken from the Okanogan River, although 70 Acrocheilus were c o l l e c t e d . Patten (1960) reports the c o l l e c t i o n of 11 hybrids and 4,260 Acrocheilus from the Yakima River. Although he says that t h i s i s a high incidence of hybridization, the r e l a t i v e abundance of 0.3 percent i s the lowest figure which was obtained for any hybrid l o c a l i t y . Two rotenone c o l l e c t i o n s made i n Wolfe Lake i n 1965 contained one year old cyprinids. At t h i s stage p o s i t i v e i d e n t i f i c a t i o n of the hybrids was possible, and pooled abundance of hybrids for the two samples was 3.45 per cent r e l a t i v e to Acrocheilus. The decrease i n abundance of hybrids between t h i s sample and the adult f i s h does not necessarily suggest s e l e c t i o n against hybrids. I t was mentioned previously that the samples of juveniles were Table I I I Species compos i t ion o f poo led ca tch from M i s s e z u l a Lake and Wolfe Lake M i s s e z u l a Lake, Wolfe Lake, 1965 P tychoche i l u s A c r o c h e i l u s P t y c h o c h e i l u s x A c r o c h e i l u s H y b r i d s M y l o c h e i l u s M y l o c h e i l u s x P t y c h o c h e i l u s H y b r i d s R icha rdson ius T o t a l 1965 A d u l t F i s h Number Percent 246 19.63 A d u l t F i s h Wolfe Lake, 1965 J u v e n i l e s 96 7.66 14 1.12 893 71.27 4 0.32 occurs bu t not counted 1,253 100.00 (E.E.Moodie , per. comm.) ' Number Percent Number Percen t occurs bu t not counted 229 occurs bu t not counted may not occur occurs but not counted 232 98.69 1.31 57 58 2 373 may not occur 125 9.27 9.43 0.33 60.65 100.00 615 20.33 100.02 P t y c h o c h e i l u s x / A c r p c h e A c r o c h e i l u s ' P t y c h o c h e i l u s x / p t y c h o c h e i l u s A c r o c h e i l u s 14.58 5.69 1.31 3.45 3.51 40 more r e s t r i c t e d both in time period and area covered. Also, the juvenile and adult samples represent d i f f e r e n t year classes, and the r e l a t i v e abundance would not be comparable unless i t could be shown that hybrids are produced at the same rate every year, and that environmental factors causing mortality had operated at the same i n t e n s i t y over the span of years separating the adults sampled from the juveniles. G. F e r t i l i t y and V i a b i l i t y During the spawning season i n 1964, a l l eggs col l e c t e d were returned to Vancouver for hatching. Eggs were taken i n l o t s of about 300 each, and sur v i v a l to hatching was highly v a r i a b l e . In general, s u r v i v a l was more dependent upon the in d i v i d u a l female which was the source of eggs than i t was on the cross which was made. One female chiselmouth i n p a r t i c u l a r yielded 90 per cent hatching success i n crosses both with male chiselmouth and male squawfish. Several l o t s of eggs from both chiselmouth and squawfish, representing both intra-species crosses and hybrid crosses, were-observed during the f i r s t four days of development. In a l l cases, rearing temperature was 20°C. At t h i s temperature, development appeared normal for a l l crosses. By the beginning of the t h i r d day, 15 41 somites were v i s i b l e , and by 84 hours (3h days) the blastopore had closed. In other l o t s which were reared through to hatching, eye pigmentation was apparent by the tenth day, and hatching was complete by 14 - 17 days. Hybrids with mature gametes were infrequent. During the 1964 season, a s i n g l e male was c o l l e c t e d . This i n d i v i d u a l d i f f e r e d i n some respects from a t y p i c a l hybrid morphology, and was the male parent of the backcross o f f -spring discussed previously. Three t y p i c a l hybrids,, with free gametes, two males and one female, were c o l l e c t e d during the 1965 season. Each of the two males was used to f e r t i l i z e both chiselmouth and squawfish eggs. Quali t a t i v e -l y , the m i l t from these males was translucent, rather than the opaque white t y p i c a l for parent species males, having a "watery" appearance. In both cases, about 40% (range 30-70%) of the eggs showed malformed blas t o d i s c s a f t e r 6 hours incubation at 18°C. After 24 hours incubation, 50% -70% of the eggs f e r t i l i z e d by the hybrid males were dead. The remaining l i v e embryos appeared normal, but a l l died within four days of f e r t i l i z a t i o n . I t should be noted, however, that at t h i s time nearly a l l the eggs of i n t r a -species crosses being reared also died. While the mortality of the f i r s t 24 hours cannot be explained by environmental causes, the l a t e r mortality at 3-4 days was probably due to i n s u f f i c i e n t oxygen i n the water, and affected a l l the eggs being reared at t h i s time. 42 The female hybrid yielded eight l o t s of about 200 apparently normal eggs each. Four of these l o t s were f e r t i l i z e d with chiselmouth sperm and four with squawfish sperm. A l l but one of these l o t s began to swell and turn cl e a r on immersion i n water a f t e r f e r t i l i z a t i o n , and were obviously dead a f t e r 10 minutes. The single l o t which did not behave i n t h i s way had about 50% obviously dead eggs af t e r 6 hours and 100% mortality i n 24 hours. Although swelling did not follow immersion as r a p i d l y i n t h i s l o t , the dead eggs appeared to have suffered the same fate over a longer period of time, a l l appearing swollen and clear when dead rather than opaque white which was t y p i c a l of dead eggs of the parent species. During the 1964 season, the duration of sperm m o t i l i t y was examined for both parent species. Sperm became motile when placed into water. At 18°C., m o t i l i t y Was l o s t at the same rate i n both species. From 15-20 seconds a f t e r immersion, there appeared to be no reduction in m o t i l i t y . By 30 seconds, 50% of the sperm was no longer moving, and by 50-60 seconds, there was no remaining m o t i l i t y . A gamete l i f e of 30 seconds would allow viable sperm to d r i f t from 18.3 - 54.9 M. i n the water v e l o c i t i e s observed i n Wolfe Greek. This equals or exceeds the dimensions of a l l the pools and runs i n which both species were observed so that accidental f e r t i l i z a t i o n by d r i f t i n g 43 sperm i s a d i s t i n c t p o s s i b i l i t y i n producing hybrids i n Wolfe Creek. D. Cytology The d i p l o i d number most frequently recorded for Acrocheilus i s 46, and for Ptychocheilus, 52. (Table IV). In both cases, these were also the highest counts observed. J.D. McPhail (per., comm.) observed counts of both 50 and 52 for Chehalis and Columbia River Ptychocheilus. On the basis of these counts, one would expect F^ hybrids to have a d i p l o i d number equal to the sum of the haploid numbers of the two parents, or 49. In fact, counts of 49 and 50 were most commonly observed, and appeared to depend upon which hybrid r e c i p r o c a l was involved, the female Ptychocheilus r e c i p r o c a l showing counts of 50 and the female Acrocheilus r e c i p r o c a l giving counts of 49. y In constructing karyograms of the best nuclei, there was close agreement between d i f f e r e n t n u c l e i from the same species, such that, when chromosomes were ranked in descending order at t o t a l length, the morphology of the l chromosome at any given p o s i t i o n in the rank was l i k e the one from the same p o s i t i o n i n other n u c l e i of the same species, A t o t a l of s i x Ptychocheilus and four Acrocheilus n u c l e i were s u f f i c i e n t l y free of chromosome overlaps to permit construction of karyograms (Figs. 11, 44 F i g . 11. Colchicine treated m i t o t i c metaphase from cultured t e s t i s of Acrocheilus alutaceus. Probable p a i r i n g of chromosomes i s shown below photograph of nucleus. 2N = 46. J \ ( ft 11 11 II If t l 1 2 3 4 5 6 7 8 It II II 1 < H i i l l II 9 1 0 11 1 2 1 3 1 4 1 5 1 6 ti 1 7 II 1 8 1 9 ( I 2 0 IS 2 1 II 2 2 3 ( X - Y ? ) Table IV Frequency of d i p l o i d chromosome numbers for wild parental species and reared F, Hybrids Species 41 42 Observed Chromosome Numbers 43 44 45 46 47 48 49 50 51 52 Reared Hybrids Acrocheilus  alutaceus ''Female Acrocheilus r e c i p r o c a l Female Ptychocheilus r e c i p r o c a l Ptychocheilus  oregonensis 6 2 3 8 2 3 1 10 45. 46 12 and 13). If the observed number of chromosomes was grossly above or below the actual number, one would expect to fi n d cases of t r i p l e t s or unpaired chromosomes, but none occurred. A l l the material from the parent species was obtained from males. The asymmetrical p a i r of chromosomes (See Figs. 11 and 12) pos s i b l y represent sex chromosomes. Again, McPhail has observed the same asymmetrical p a i r in Chehalis River Ptychocheilus. There i s no evidence, however, that t h i s p a i r i s heterochromatic, and data on th e i r meiotic behavior are lacking. Among the larger chromosomes, i n which the morphology i s d i s t i n c t i v e , close agreement can be seen between Acrocheilus and Ptychocheilus. One notable d i f -ference i s i n the smaller of the two possible sex chromosomes, which i s a very small metacentric in Ptychocheilus and a larger submetacentric i n Acrocheilus. Arm numbers calculated from the karyograms are given in Table V. From t h i s i t cannot be shown that any of the Acrocheilus chromosomes represent c e n t r i c fusions of two Ptychocheilus chromosomes. Also, there i s no higher proportion of nearly symmetrical chromosomes i n Acrocheilus than in Ptychocheilus, which could indicate translocations. I t can only be concluded that Ptychocheilus has more chromatin. 4 7 F i g . 12, Colchicine treated m i t o t i c metaphase from Ptychocheilus oregonensis cultured t e s t i s . Probable p a i r i n g of chromosomes i s shown below photograph of nucleus. 2N = 52. # H tl tt u 11 ui t i 1 2 3 4 5 6 7 8 If U ft I t i i *« i t n 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 t i M at t t » i t t i i i 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 A^ it 2 5 2 6 ( X - Y ? ) 4 8 P i g . 13. Colchicine treated m i t o t i c metaphases from branchial epithelium of reared F i hybrids. AboveJ female Acrocheilus r e c i p r o c a l ; 2N = 50. Belows female Ptychocheilus r e c i p r o c a l : 2N = 49. 49 Table V Chromosome arm numbers observed for wild parental species from Wolfe Lake and the Chehalis River Species Acrocheilus  alutaceus (Wolfe Lake) Observed Arm Number Number of Nuclei 88 4 Ptychocheilus  oregonensis (Wolfe Lake) 98 P ty cho ch e i l u s  oregonensis (Chehalis River, McPhail, per. comm.) 96 50 In the absence o f da ta on m e i o t i c behav io r o f the h y b r i d chromosome the amount o f homology between the two spec ies can o n l y be i n f e r r e d . Because o f the g e n e r a l l y c l o s e resemblance i n chromosome morphology, i t i s p o s s i b l e t ha t there i s a h i g h degree o f homology, w i t h the three e x t r a P t y c h o c h e i l u s chromosomes a c t i n g as u n i v a l e n t s . Fu r the r evidence fo r a h i g h degree o f homology i s the presence o f probable backcrosses i n the p o p u l a t i o n . I f there were a l a r g e number o f non homologous chromosomes, then p a i r i n g o f the chromosomes d u r i n g the prophase I o f me ios i s i n F-^  h y b r i d s would be comple te ly b l o c k e d , and the h y b r i d s would be comple te ly s t e r i l e . S ince some F^ h y b r i d s were observed to have a t l e a s t p a r t i a l f e r t i l i t y , and s i n c e presumed backcrosses were c o l l e c t e d , a h i g h degree o f chromosomal homology must be assumed. E . Spawning H a b i t a t and Behav io r A l l the da ta on n a t u r a l spawning areas o r i g i n a t e from M i s s e z u l a and Wolfe Lakes . The l o c a l i t i e s where f i s h i n spawning c o n d i t i o n were observed or taken are shown i n F igu res 2 and 3 . A c r o c h e i l u s w i t h f ree gametes were f i r s t taken i n g i l l n e t samples on June 9 d u r i n g the 1965 season. Dur ing the 1964 season, they were f i r s t taken on June 12, bu t may have been there e a r l i e r . Surface water temperature i n a l l 51 r eg ions o f the l a k e was 18°C. when spawning f i s h o f bo th spec ies were f i r s t observed . Dur ing c l e a r weather, mature A c r o c h e i l u s appeared to move i n t o the o u t l e t r e g i o n each n i g h t , and remain there , s h e l t e r i n g under b rush and logs d u r i n g the day. Cloudy weather appeared to stop the movement i n t o the o u t l e t and cause those f i s h which were there to move back i n t o the l a k e . Observa t ions by d i v i n g d u r i n g the 1964 season showed tha t a schoo l o f A c r o c h e i l u s es t imated to c o n t a i n from 20 to 50 f i s h was presen t i n the o u t l e t on s e v e r a l c l e a r days i n s u c c e s s i o n . A few P t y c h o c h e i l u s t e n t a t i v e l y i d e n t i f i e d as males because o f t h e i r s m a l l s i z e and the presence o f w e l l - d e v e l o p e d t u b u r c l e s on the head and p e c t o r a l f i n s , were inc luded: i n the s c h o o l . The bes t v i s u a l es t imate o f P t y c h o c h e i l u s abundance was a count o f s i x i n d i v i d u a l s i n a s c h o o l es t imated to c o n t a i n about 50 A c r o c h e i l u s and one h y b r i d . Male P t y c h o c h e i l u s were a l s o taken i n the g i l l n e t samples from the o u t l e t r e g i o n a t the r a t e o f one to about ten A c r o c h e i l u s . Dur ing bo th seasons, o n l y three s e x u a l l y mature female P t y c h o c h e i l u s were taken from the o u t l e t . P t y c h o c h e i l u s i n M i s s e z u l a Lake were observed to spawn i n an area along; the west shore o f the l a k e , about 0.8 Km. no r th o f the o u t l e t r e g i o n ( F i g . 2 ) . S e x u a l l y mature male P t y c h o c h e i l u s were a l s o found i n the o u t l e t o f M i s s e z u l a Lake a t the same time tha t A c r o c h e i l u s began to 52 appear, a l though spawning i n P t y c h o c h e i l u s was not observed u n t i l June 21 d u r i n g the 1965 season. T h i s , however, was p robab ly due to d i f f i c u l t y i n f i n d i n g P t y c h o c h e i l u s spawning l o c a l i t i e s , s i n c e a mature female was taken i n the o u t l e t on June 12 d u r i n g the 1964 season and on June 15 d u r i n g the 1965 season. Spawning was observed on four days d u r i n g the 1965 season, each time a t dusk, from 7.30 p .m. to 10 p .m. A dense mass o f f i s h , appa ren t ly males, j udg ing from t h e i r s i z e , occupied the areai immediate ly o f f s h o r e . Some o f the f i s h c l o s e s t to shore were mos t ly out o f the wa te r . The dimensions o f the aggrega t ion were about 3.1 M. from inshore to o f f sho re e x t r e m i t y and about 4 .6 M. p a r a l l e l to the shore . In the deeper water , o f f sho re from the aggrega t ion o f males, l a r g e r f i s h , almost c e r t a i n l y females, were swimming around the p e r i p h e r y o f the agg rega t ion . Females appa ren t ly entered, the aggrega t ion near the shore, and spawning took p l a c e i n water too sha l low to cover the f i s h , accompanied by much s p l a s h i n g by the males . In a few cases , the female was s u f f i c i e n t l y v i s i b l e to see her body v i b r a t i n g , presumably d u r i n g emiss ion o f the eggs. On one o c c a s i o n the aggrega t ion d i s p e r s e d due to d i s tu rbance bu t re-formed i n about 30 minutes . A t n i g h t f a l l , the s c h o o l d i s p e r s e d , and no f u r t h e r a c t i v i t y was heard or seen when obse rva t ions were extended to one hour a f t e r n i g h t f a l l . No spawning a c t i v i t y was observed d u r i n g the e a r l y morning, j u s t a f t e r dawn. 52 appear, a l though spawning i n P t y c h o c h e i l u s was not observed u n t i l June 21 d u r i n g the 1965 season. T h i s , however, was p robab ly due to d i f f i c u l t y i n f i n d i n g P t y c h o c h e i l u s spawning l o c a l i t i e s , s i n c e a mature female was taken i n the o u t l e t on June 12 d u r i n g the 1964 season and on June 15 d u r i n g the 1965 season. Spawning was observed on four days d u r i n g the 1965 season, each time a t dusk, from 7.30 p .m. to 10 p .m. A dense mass o f f i s h , appa ren t ly males, j udg ing from t h e i r s i z e , occupied the area immediately o f f s h o r e . Some o f the f i s h c l o s e s t t o shore were mos t ly out o f the wa te r . The dimensions o f the aggregat ion were about 3.1 M. from inshore to o f f sho re e x t r e m i t y and about 4 .6 M. p a r a l l e l to the shore . In the deeper water , o f f sho re from the aggrega t ion o f males, l a r g e r f i s h , almost c e r t a i n l y females, were swimming around the p e r i p h e r y o f the agg rega t ion . Females appa ren t ly en te red the aggrega t ion near the shore, and spawning took p l a c e i n water too sha l low to cover the f i s h , accompanied by much s p l a s h i n g by the males . In a few cases , the female was s u f f i c i e n t l y v i s i b l e to see her body v i b r a t i n g , presumably d u r i n g emiss ion o f the eggs. On one o c c a s i o n the aggrega t ion d i s p e r s e d due to d i s tu rbance bu t re-formed i n about 30 minutes . A t n i g h t f a l l , the s choo l d i s p e r s e d , and no f u r t h e r a c t i v i t y was heard or seen when obse rva t ions were extended to one hour a f t e r n i g h t f a l l . No spawning a c t i v i t y was observed d u r i n g the e a r l y morning, j u s t a f t e r dawn. 53 Examinat ion o f the, spawning l o c a l i t y r evea led l a r g e numbers o f eggs, a l l ve ry c l o s e to shore, i n l e s s than 15 cm. o f wa te r . The area o f egg d e p o s i t i o n extended fo r about 3.6 M. a long the shore . G i l l n e t c o l l e c t i o n s from t h i s l o c a l i t y y i e l d e d l a r g e numbers o f r i p e male P t y c h o c h e i l u s and s m a l l e r numbers o f females i n n e a r l y mature, r i p e , or r e c e n t l y spent c o n d i t i o n . Only one A c r o c h e i l u s , a spent male, was c o l l e c t e d i n t h i s area near the end o f the spawning season ( J u l y 12 ) . P t y c h o c h e i l u s p robab ly spawns i n o ther areas o f the l a k e , bu t these were not l o c a t e d d u r i n g the course o f the s tudy . I t cannot be shown c o n c l u s i v e l y t ha t A c r o c h e i l u s spawns o n l y i n the- o u t l e t r e g i o n . I t i s not a common f i s h i n the l a k e , however, and i t s h i g h r a t e o f occurrence i n the o u t l e t r e g i o n d u r i n g the spawning season would seem to be s u f f i c i e n t t o account f o r the spawning o f the e n t i r e l a k e p o p u l a t i o n . In Wolfe Lake, bo th A c r o c h e i l u s and P t y c h o c h e i l u s u t i l i z e d the i n l e t stream, Wolfe Greek, fo r spawning. Underwater obse rva t ions showed A c r o c h e i l u s and P t y c h o c h e i l u s to be abundant i n the deeper poo l s and runs i n the lower h a l f o f the s t ream's l e n g t h . A l a r g e p o r t i o n o f the remain ing d i s t a n c e upstream to I z z i t ' s Lake c o n s i s t e d o f s t r a i g h t runs w i t h l i t t l e cover and almost no f i s h . For about 91.4 meters below I z z i t ' s Lake, poo l s were p resen t , 54 occupied again by Acrocheilus and Ptychocheilus. These f i s h may, however, have originated from I z z i t ' s Lake rather than Wolfe Lake. Unlike Missezula Lake, there was no s p a t i a l segregation of Acrocheilus and Ptychocheilus i n Wolfe Creek. Both were abundant wherever conditions were suitable. Also, the Acrocheilus appeared to form groups which moved together, independent of the Ptychocheilus. Non-sexual interactions, p r i m a r i l y nudging, chasing, and grouping together, appeared to be confined to within each species and no i n t e r s p e c i f i c acts were observed. This i s i n contrast to observations i n the ou t l e t of Missezula Lake, where the Ptychocheilus males a c t i v e l y oriented themselves with, and moved about with Acrocheilus. Spawning was not observed i n Wolfe Creek, but eggs co l l e c t e d from two of the runs where both parent species were present were reared i n the laboratory. The f r y produced were mostly Ptychocheilus, but a number of Acrocheilus were also present. 5 5 DISCUSSION A. Origin of the Wild Intermediates Four l i n e s of evidence are available which indicate that the wild intermediates are the r e s u l t of hybr i d i z a t i o n between Ptychocheilus and Acrocheilus, and that most of them are F^ hybrids. F i r s t , the comparative morphology of the wild intermediates and the parental species shows that the intermediates, for the most part, l i e between the values for the parents in the characters examined. There would appear to be some dominance of squawfish features, however, since the intermediates lack the keratinized margin of the lower jaw, t y p i c a l l y show two teeth on the outer, pharyngeal tooth row, and lack the c o i j i n g t y p i c a l of the chiselmouth gut. Second, with the exception of the disagreement i n outer row pharyngeal tooth counts i n the female Acrocheilus reciprocals, the reared F^ hybrids are sim i l a r i n a l l respects to the wild intermediates. Third, the o f f s p r i n g of the backcross experiment, i n which the aberrant male from Missezula Lake was crossed to a female Acrocheilus, show a pharyngeal tooth count segregation that can only be explained by assuming that the male had chiselmouth genes. This male shared with t y p i c a l intermediates the dark peritoneum and the long anterior f o l d of the i n t e s t i n e . I t d i f f e r e d in having a 56 s t r a i g h t , r a t h e r than decurved mouth and i n a lower s c a l e count . I t d i f f e r e d from presumed M y l o c h e i l u s x P t y c h o c h e i l u s h y b r i d s from M i s s e z u l a Lake i n t ha t the l a t t e r have a s i l v e r y per i toneum, a sho r t a n t e r i o r f o l d o f the i n t e s t i n e and, l i k e M y l o c h e i l u s , the v e n t r a l two pharyngeal t ee th o f the inne r row are sho r t and b l u n t r a t h e r than p o i n t e d as i n A c r o c h e i l u s , P t y c h o c h e i l u s , and i n the M i s s e z u l a Lake aber ran t male. Th i s male a l s o d i f f e r e d from the reared h y b r i d s between P t y c h o c h e i l u s and R icha rdson ius i n t ha t the l a t t e r show a s i l v e r y per i toneum w i t h d i f f u s e melanophores, a h i g h ana l r ay count (11, as opposed to 7, 8 o r 9 fo r P t y c h o c h e i l u s and A c r o c h e i l u s i n M i s s e z u l a Lake) and i n the more o b l i q u e mouth i n P t y c h o c h e i l u s x R icha rdson ius h y b r i d s . T h i s l eaves open o n l y two p o s s i b i l i t i e s ; e i t h e r the aber ran t male was an unusual P t y c h o c h e i l u s , o r was o f h y b r i d o r i g i n . I t has been shown tha t rea red F^ h y b r i d s and w i l d i n t e r -mediates always have a t l e a s t one ou te r row pharyngeal t o o t h . I f the male i n ques t i on were an aber ran t P t y c h o c h e i l u s , then the same r e s u l t would be expected upon c r o s s i n g him to a female A c r o c h e i l u s . The f a c t t h a t 62 per cent o f the o f f s p r i n g showed a 0-0 count excludes the p o s s i b i l i t y t ha t he was an aber ran t P t y c h o c h e i l u s , l e a v i n g o n l y h y b r i d o r i g i n as an e x p l a n a t i o n . Fou r th , the w i l d in te rmedia tes are p a r t i a l l y s t e r i l e . Of those which develop sex p roduc t s , the females appear to produce i n v i a b l e eggs and about 40 per cent o f 5 7 parental eggs f e r t i l i z e d with sperm from intermediate males show abnormal cleavage and die. These observations exclude the p o s s i b i l i t y that the wild intermediates might be a rare and h i t h e r t o unrecognized species which reproduces i t s e l f . The p r o b a b i l i t y that most of the wild hybrids are P-j^ s . i s indicated by t h e i r agreement i n morphology with reared F-^s, and the observed high degree of s t e r i l i t y in wild hybrids. Because of t h e i r low abundance and high degree of s t e r i l i t y , the chances of hybrid to hybrid matings i s extremely low (Hubbs, 1 9 5 5 ) i f not absent altogether i n view of the apparent t o t a l i n v l a b i l i t y of hybrid eggs. I f other than F ^ hybrids are to occur, they must almost c e r t a i n l y be the r e s u l t of backcrossing to one or the other parent species. In fact, the four wild intermediates of aberrant morphology seem to suggest t h i s type of o r i g i n . The two from Missezula Lake might be interpreted as F ^ x Ptychocheilus backcrosses, while the two Wolfe Lake examples might be F]_ x Acrocheilus backcrosses. I t seems certai n that the Missezula Lake individuals are of hybrid o r i g i n , from the argument outlined above. The differences between these individuals and t y p i c a l hybrids are i n the more Ptychocheilus - 1ike values for some characters. Because of t h i s , FJL to Ptychocheilus backcrosses would seem to be the most l o g i c a l explanation of t h e i r o r i g i n . The Wolfe Lake aberrants show a morphology 58 i n t e rmed ia t e between an and A c r o c h e i l u s , which would suggest b a c k c r o s s i n g w i t h A c r o c h e i l u s as t h e i r o r i g i n . The evidence i n t h i s case i s not as d i r e c t , however. A c r o c h e i l u s i s known to h y b r i d i z e o n l y w i t h P t y c h o c h e i l u s , bu t M y l o c h e i l u s and R icha rdson ius are a l s o i n the same area d u r i n g the spawning p e r i o d . An A c r o c h e i l u s male x R icha rdson ius female c ross was made a t Wolfe l a k e u s i n g 10 R icha rdson ius eggs. One i n d i v i d u a l s u r v i v e d to a s i z e a t which s ca l e s were formed, and i t s morphology i s d i f f e r e n t from the Wolfe Lake abe r r an t s . The p o s s i b l e morphology o f A c r o c h e i l u s x M y l o c h e i l u s i s a s p e c u l a t i v e mat te r . In the two A c r o c h e i l u s h y b r i d c rosses which were made a r t i f i c i a l l y however, the k e r a t i n i z e d edge on the lower jaw was t o t a l l y surpressed , as was the gut c o i l i n g . I f an A c r o c h e i l u s x M y l o c h e i l u s c ross c o u l d be expected to behave s i m i l a r l y , then the Wolfe Lake aberrants would d i f f e r s i n c e they have a k e r a t i n i z e d margin on the lower jaw and an i n f l e c t e d gut . B . Causes o f H y b r i d i z a t i o n In a l l known h y b r i d l o c a l i t i e s except M i s s e z u l a Lake, the h y b r i d s are r e l a t i v e l y r a r e . Wolfe Lake, w i t h a 1.3 per cent r e l a t i v e abundance o f h y b r i d s i s the h i g h e s t f i g u r e recorded except f o r M i s s e z u l a Lake, where the abundance o f h y b r i d s i s 13.2 per cent r e l a t i v e to A c r o c h e i l u s . The l a r g e gap between M i s s e z u l a Lake and a l l the o ther 59 h y b r i d l o c a l i t i e s i n r e l a t i v e abundance o f h y b r i d s suggests t ha t two d i f f e r e n t mechanisms may be o p e r a t i n g to cause hybr i d i z a t i o n . In M i s s e z u l a Lake, both spec ies spawn i n the l a k e . A c r o c h e i l u s uses the o u t l e t narrows above the dam, apparen t ly to the e x c l u s i o n o f a l l o the r areas , and P t y c h o c h e i l u s spawns i n the main body o f the l a k e . Some s e x u a l l y mature P t y c h o c h e i l u s are found i n the o u t l e t r e g i o n , away from t h e i r observed spawning a rea . Because o f t h i s , the schoo l i n the o u t l e t i s mixed, c o n t a i n i n g mos t ly s e x u a l l y mature A c r o c h e i l u s and a few, mos t l y mature male P t y c h o c h e i l u s . P t y c h o c h e i l u s was observed to be a group spawner, w i t h a number o f males spawning w i t h one or a few females a t the same t ime . S c h u l t z (1935) observed a s i m i l a r spawning h a b i t i n M y l o c h e i i u s , and i n Wolfe Creek, R icha rdson ius was observed to spawn i n ' l a r g e groups w i t h no apparent p a i r fo rma t ion . A c r o c h e i l u s was not seen spawning, bu t the p a t t e r n i s presumably s i m i l a r to t ha t observed f o r the o ther th ree r e l a t e d genera ." I f group spawning i s assumed f o r A c r o c h e i l u s , then h y b r i d i z a t i o n may a r i s e i n one or both o f the f o l l o w -i n g ways. F i r s t , s i n c e there are few male P t y c h o c h e i l u s s c h o o l i n g w i t h the A c r o c h e i l u s i n the o u t l e t , these might p a r t i c i p a t e i n f e r t i l i z a t i o n o f the eggs o f an A c r o c h e i l u s female. Second, s i n c e female P t y c h o c h e i l u s w i t h f ree eggs occur o c c a s i o n a l l y i n the o u t l e t , they may a l s o spawn t h e r e . 60 In the case o f the M i s s e z u l a Lake o u t l e t , the o n l y male P t y c h o c h e i l u s a v a i l a b l e are s c h o o l i n g w i t h the A c r o c h e i l u s , and i t would seem, on the b a s i s o f r e l a t i v e numbers o f the two spec ies p resen t i n the o u t l e t , t ha t a spawning female P t y c h o c h e i l u s would more l i k e l y h y b r i d i z e than spawn w i t h a male o f her own s p e c i e s . I f the d i f f e r e n c e i n pharyngeal too th counts between reared female A c r o c h e i l u s and female P t y c h o c h e i l u s r e c i p r o c a l c rosses i s c o n s i s t e n t i t i n d i c a t e s t ha t the b u l k o f w i l d h y b r i d s are from P tychoche i 1us eggs. P o s s i b l y , h y b r i d i z - ; a t i o n v i a female P t y c h o c h e i l u s i s the b e s t e x p l a n a t i o n fo r the h i g h r e l a t i v e abundance o f h y b r i d s i n M i s s e z u l a Lake . I f h y b r i d i z a t i o n was o c c u r i n g o n l y through mixed males f e r t i l i z i n g female A c r o c h e i l u s , the n u m e r i c a l l y l e s s abundant Ptychochei1us males c o u l d f e r t i l i z e o n l y a s m a l l p r o p o r t i o n o f the eggs o f any female, and a d u l t h y b r i d s would be l e s s abundant than observed. On the o ther hand, a female P t y c h o c h e i l u s i n the same s c h o o l o f f i s h would l o s e most o f her eggs t o - h y b r i d i z a t i o n because o f the g rea t e r abundance o f male A c r o c h e i l u s . Hubbs, Walker and Johnson (1943), and Hubbs, Hubbs and Johnson (1943) r e p o r t cases where i n t e r s p e c i f i c h y b r i d i z a t i o n o f a t h e r i n i d s and catos tomids r e s p e c t i v e l y are p robab ly due to a d i s p r o p o r t i o n a t e r e l a t i v e abundance o f the p a r e n t a l s p e c i e s . Mayr (1963) a l s o c i t e s cases i n b i r d s where zones o f con tac t e s t a b l i s h e d on the p e r i p h e r y 61 o f the range o f one p a r e n t a l form can l e a d to h y b r i d i z a t i o n . Th i s a l s o a p p l i e s to r a r e s t r a y s i n n o r m a l l y segregated r e p r o d u c t i v e groups w i t h i n a s m a l l a rea . The s t r ays , be ing w i t h i n the mating area o f the o ther s p e c i e s , w i l l be unable to f i n d c o n s p e c i f i c mates and w i l l h y b r i d i z e (Mayr, 1963). In Wolfe Creek, the c i rcumstances are q u i t e d i f f e r e n t . Here there i s no observable seg rega t ion o f s e x u a l l y mature A c r o c h e i l u s and P t y c h o c h e i l u s . Both spec ies are concent ra ted i n the deeper poo l s and runs, u s i n g what-ever top cover i s a v a i l a b l e . Spawning was not observed i n Wolfe Creek, bu t bo th spec ies must spawn i n the same areas where the aggregat ions were seen, s i n c e eggs taken from those areas and reared proved to be long to bo th s p e c i e s . S ince the r e l a t i v e abundance o f a d u l t h y b r i d s i s much lower i n Wolfe Lake than i n M i s s e z u l a Lake, spec ies r e c o g n i t i o n must take p l a c e , s i n c e there i s no s p a t i a l s e g r e g a t i o n . The r e l a t i v e abundance o f A c r o c h e i l u s and P t y c h o c h e i l u s as es t imated from v i s u a l counts o f s e x u a l l y mature f i s h i n Wolfe Creek, i s n e a r l y e q u a l . In c o n t r a s t , i n the o u t l e t o f M i s s e z u l a Lake, P t y c h o c h e i l u s i s r a r e r e l a t i v e to A c r o c h e i l u s , and schoo l s w i t h A c r o c h e i l u s . The o n l y obse rva t ions made i n Wolfe Creek which are a p p l i c a b l e to the problem o f mate s e l e c t i o n i n the two spec ies are tha t , i n the aggrega t ions , the A c r o c h e i l u s seem to c l u s t e r toge ther , r a t h e r than b e i n g s c a t t e r e d u n i f o r m l y among the P t y c h o c h e i l u s . A l s o , the o n l y i n t e r a c t i o n s 62 which were observed were intraspecif ic , suggesting that species recognition is operating, at least in non-sexual interactions. If, in fact, species recognition and segretation are operative when, relative abundance is more nearly equal, then chance encounters of dr i f t ing gametes may account for the level of hybridization observed. From experiments on sperm of Missezula Lake Acrocheilus and Ptychocheilus i t would appear that the sperm can be in contact with water for 15 to 20 seconds before moti l i ty begins to decline. At the water velocit ies measured in Wolfe Creek in areas where sexually mature fish were observed, sperm could d r i f t from 4.6 to 36.3 meters, depending on the water velocity before their moti l i ty began to decline. Since the two species were observed to be closer together than this , and since eggs of both were taken from the same area, chance f er t i l i za t ion probably does occur. This could be a general phenomenon, since the spawning habitat used by the two species is similar in most respects. Hubbs (1957, 1960 and 1961) has studied duration of sperm function and gamate compatibility as related to interspecif ic hybrid formation. The delay times he observed before f er t i l i za t ion percentages began to decline are comparable to the delay times observed for Acrocheilus and Ptychocheilus before moti l i ty declined sharply. 6 3 C. Consequences o f H y b r i d i z a t i o n Swamping of e i t h e r p a r e n t a l form has not occur red , even where the r a t e o f h y b r i d i z a t i o n i s as h i g h as i n M i s s e z u l a Lake . I f i n t r o g r e s s i o n has taken p l a c e , i t i s not r evea led i n the morphology o f e i t h e r paren t s p e c i e s . The w i l d p o p u l a t i o n s appear to c o n s i s t o f d i s t i n c t and una l t e r ed parent spec i e s , w i t h in t e rmed ia te s , mos t ly F i h y b r i d s appear ing i n s c a t t e r e d l o c a l i t i e s throughout the sympatr ic range o f the paren t s p e c i e s . S ince the F^ h y b r i d s are p a r t i a l l y f e r t i l e , and r a r e backcrosses are known, b a r r i e r s o ther than s t e r i l i t y must p revent gene f low between the paren t s p e c i e s . The low abundance and p a r t i a l s t e r i l i t y o f the h y b r i d s , combined w i t h the s t rong morpho log ica l and e c o l o g i c a l d ivergence o f the paren t spec i e s , c o u l d operate to l i m i t the r a t e a t which b a c k c r o s s i n g takes p l a c e , and a l s o , to r e s t r i c t s e v e r e l y the s u r v i v a l o f backcross o f f s p r i n g because o f t h e i r unbalanced and non-adapt ive genotypes. Swamping or i n t r o g r e s s i o n can take p l a c e o n l y i f the h y b r i d s themselves are a t a s e l e c t i v e advantage, or i f c e r t a i n gene complexes o f one paren t are a t a s e l e c t i v e advantage to t h e i r a l t e r n a t i v e s i n the o ther parent (Anderson, 1 9 5 3 , Mayr, 1 9 6 3 , B ige low, 1 9 6 5 ) . In order fo r F-L h y b r i d s to s u r v i v e a t a l l , there must be a s u i t a b l e environment open to them. For h y b r i d genera t ions beyond 64 the F-^, or fo r backc rosses , recombina t ion w i l l cause much grea te r v a r i a b i l i t y than i n F i h y b r i d s , w i t h the r e s u l t t ha t many recombinants w i l l be i n v i a b l e o r p o o r l y adapted. In organisms which are w i d e l y d i v e r g e n t , the number o f p o s s i b l e adap t ive recombinants would be o n l y a sma l l f r a c t i o n o f the t o t a l number o f p o s s i b l e recombinants . T h i s would s e v e r e l y r e s t r i c t the s u r v i v a l . o f backcross o f f s p r i n g , or o f h y b r i d genera t ions beyond the F^ , though s u r v i v a l o f F^s may be good. S ince the F-^  h y b r i d s are n u m e r i c a l l y r a r e , they w i l l p robab ly backcross to one o f the paren t spec i e s , r a t h e r than mating w i t h one another (Hubbs, 1 9 5 5 ) . I t has been shown tha t P t y c h o c h e i l u s i s a mass-spawner, and t h a t A c r o c h e i l u s p robab ly i s . A l s o , the evidence i n d i c a t e s i n female F^ h y b r i d s , any eggs which may be produced are i n v i a b l e . The o n l y channel open f o r gene f low, then, would be the male F^s , o f which o n l y 3 out o f 20 examined had f ree gametes. In order fo r a male F^ t o f e r t i l i z e any p a r e n t a l eggs, he would have to compete w i t h a r e l a t i v e l y l a r g e number o f A c r o c h e i l u s o r P t y c h o c h e i l u s males which were spawning w i t h the same female s i m u l t a n e o u s l y . A t b e s t , then, a male h y b r i d c o u l d f e r t i l i z e o n l y a s m a l l p o r t i o n o f the eggs o f a g iven female. From the evidence g i v e n , about 40 per cent o f these eggs would d i e be fo re c leavage was complete . I f a l l the remainder hatched, there would s t i l l be heavy m o r t a l i t y i n the o f f s p r i n g fo r 65 the reasons outlined above. Only one backcross, the second Missezula Lake male, had free gametes. The f i r s t Missezula Lake backcross was col l e c t e d on 26th July, or about two weeks afte r spawning in both parent species was finished in the two seasons of observation, and appeared to be unspawned. Neither of the Wolfe Lake backcrosses had free eggs, although the ovaries of both appeared grossly normal and well developed. The second Missezula Lake backcross male produced sperm with high f e r t i l i t y , but t h i s does not mean that a l l backcrosses would necessarily be f e r t i l e . While the lack of free eggs i n Wolfe Lake backcross females might mean only that they had not yet completed maturation, the unspawned condition of the f i r s t Missezula Lake male backcross i s p o s i t i v e evidence that he was somehow barred from reproduction. Post-spawning samples of both parent species a l l had spent gonads. The presence of the w e l l -developed gonads i n t h i s backcross i s exceptional. Apparently introgression or swamping are com-p l e t e l y blocked i n both d i r e c t i o n s . The parent species maintain themselves as d i s t i n c t and separate populations with no gene exchange, and the gametes which enter into h y b ridization are l o s t . None of the available evidence indicates whether the rate of hyb r i d i z a t i o n i s s t a t i c , or varying i n some manner. I f the gamete wastage were s i g n i f i c a n t , i s o l a t i n g 6 6 mechanisms might be expected to have developed which would prevent hybridization, but t h i s does not seem to be the case. In a l l the areas studied, Ptychocheilus population l e v e l s are high enough that wastage due to hybridization i s probably not s i g n i f i c a n t . In the case of Missezula Lake, Acrocheilus i s s u f f i c i e n t l y rare for the observed l e v e l of hybridization to a f f e c t i t s abundance. This does not imply, however, that the r a r i t y of Acrocheilus i s due to hybr i d i z a t i o n . Missezula Lake affords only marginal habitat for a bottom-feeding cypr i n i d such as Acrocheilus because of the r e s t r i c t e d l i t t o r a l area. The r e l a t i v e l y recent damming of the lake may also have affected adversely the s u r v i v a l of eggs and young, since no other stream i s availab l e for spawning. Hybridization alone, then, cannot account for the low abundance of Acrocheilus i n Missezula Lake but further drops i n the population l e v e l , i f they do occur, maybe due to gamete wastage from hy b r i d i z a t i o n . In the other areas where hybrids have been collected, the abundance of Acrocheilus i s much greater, and the incidence of hybr i d i z a t i o n much lower. Since hybrids have been taken in widely scattered l o c a l i t i e s , and were known before the Columbia River System had been modified by man, i t can be concluded that h y b r i d i z a t i o n at a low l e v e l has taken place between Acrocheilus and Ptychocheilus since the two species came into contact. 67 D. Relationship of Ptychocheilus and Acrocheilus Acrocheilus and Ptychocheilus are strongly divergent i n t h e i r morphology and ecology, but much of the evidence c o l l e c t e d during the present study indicates that they are rather c l o s e l y related g e n e t i c a l l y . Several morphological characters, e s p e c i a l l y the keratinized margin of. the lower jaw, the gut c o i l i n g , and the pharyngeal tooth counts, show p a r t i a l or complete dominance ef f e c t s i n hybrids and backcrosses. Also, the myogen electropherograms f a i l e d to show any consistent differences between the two. The best single evidence of genetic s i m i l a r i t y , however, i s the i n d i c a t i o n of p a r t i a l hybrid f e r t i l i t y and the presence of wild backcrosses. I t i s possible to produce v i a b l e F^ hybrids a r t i f i c i a l l y from parents with strongly divergent genotypes. Such hybrids can also be found i n the wild. In order for a hybrid to be f e r t i l e , however, there must be s u f f i c i e n t homology between the parental chromosome complements to allow proper p a i r i n g of the chromosomes during prophase I of meiosis. Even r e l a t i v e l y small differences can lead to p a r t i a l i n f e r t i l i t y , since f a i l u r e of chromosomes to p a i r normally w i l l cause them to behave abnormally during the metaphase and anaphase portions of the f i r s t meiotic d i v i s i o n (Sinott, Dunn and Dobzhansky, 1958; Darlington, 1958). This r e s u l t s i n the secondary gametocytes 68 receiving imperfect sets of chromosomes, and thus renders them in v i a b l e . Because of the mechanics of chromosome p a i r i n g involved i n successful meiosis, the fa c t that Acrocheilus x Ptychocheilus hybrids are capable of occasionally producing v i a b l e gametes indicates a high degree of chromosomal homology. Further evidence of genetic s i m i l a r i t y i s provided by the morphology of the chromosomes. I t has been noted e a r l i e r that i n r e a d i l y i d e n t i f i a b l e p a i r s of chromosomes, there i s close comparison i n the morphology of these p a i r s between Acrocheilus and Ptychocheilus. J.D. McPhail (per. comm.) has found that t h i s s i m i l a r i t y can also be seen i n Richardsonius and Mylocheilus chromo-somes . The available evidence, then, suggests a close genetic s i m i l a r i t y , not only between Acrocheilus and Ptychocheilus, but also these two and Richardsonius and Mylocheilus. A l l permutations of hybrids between these four genera except Acrocheilus x Mylocheilus and Acrocheilus x Richardsonius are known to occur natu r a l l y . The question of the taxonomic p o s i t i o n of Acrocheilus and Ptychocheilus i s , however, a subjective matter. Obviously, they meet a l l the c r i t e r i a of good species, and t h i s has never been questioned. Whether they should be regarded as d i s t i n c t genera i s an a r b i t r a r y decision. I f t o t a l hybrid s t e r i l i t y were the c r i t e r i o n , 6 9 they should be placed i n the same genus. Many congeneric species, however, show t o t a l hybrid s t e r i l i t y , and far less morphological and ecological divergence than do Acrocheilus and Ptychocheilus. Should these be placed in separate genera? If the c r i t e r i o n for taxonomic rank were to be recentness of divergence from a common ancestor, there i s no d i r e c t evidence which i s relevant to the problem at hand. In fact, genetic divergence, or most commonly, morphological divergence, i s used as an indicator of the time of divergence from a common ancestor. The morphological and genetic evidence, however, are often i n c o n f l i c t for the same species p a i r . A further problem i n reassessment of the generic status of Acrocheilus and Ptychocheilus i s that there are not s u f f i c i e n t data available on t h e i r degree of r e l a t i o n -ship to the many other genera of western cyprinids not considered here. If Acrocheilus and Ptychocheilus were placed i n the same genus, Richardsonius and Mylocheilus should probably also be placed with them. However, since the r e l a t i o n s h i p of these four to other genera i s unknown, the best p o s i t i o n to adopt seems to be to r e t a i n the present c l a s s i f i c a t i o n u n t i l the required data are av a i l a b l e . SUMMARY The w i l d i n t e r m e d i a t e s between A c r o c h e i l u s and P t y c h o c h e i l u s a r e o f h y b r i d o r i g i n , and most o f them a r e F-^ h y b r i d s . When r e l a t i v e abundance o f the two p a r e n t a l s p e c i e s i s d i s p r o p o r t i o n a t e , as i n M i s s e z u l a L a k e , a c t i v e i n t e r s p e c i f i c mat ing p r o b a b l y takes p l a c e . The o c c u r r e n c e o f r a r e F ^ h y b r i d s from many o t h e r l o c a l i t i e s sugges ts t h a t h a b i t a t m o d i f i c a t i o n i s no t p r e r e q u i s i t e to h y b r i d i z a t i o n . A l l known h y b r i d l o c a l i t i e s o t h e r than M i s s e z u l a Lake a r e i n s treams, and the e v i d e n c e c o l l e c t e d i n Wol fe Creek sugges ts t h a t h y b r i d i z a t i o n under u n a l t e r e d c o n d i t i o n s may be caused b y d r i f t i n g gametes meet ing b y chance . None o f the m o r p h o l o g i c a l e v i d e n c e sugges t s t h a t swamping o r i n t r o g r e s s i o n has taken p l a c e , a l t h o u g h r a r e , a b e r r a n t i n d i v i d u a l s a r e a lmost c e r t a i n l y b a c k c r o s s e s . The mechanisms which have p r e v e n t e d gene f low between the p a r e n t a l s p e c i e s a r e a h i g h degree o f s t e r i l i t y i n the F^ h y b r i d s , the n u m e r i c a l r a r i t y o f h y b r i d s , which lowers the p r o b a b i l i t y o f h y b r i d t o h y b r i d mat ing o r o f b a c k c r o s s i n g , and p r o b a b l e s e v e r e s e l e c t i o n a g a i n s t recombinants i n any 71 b a c k c r o s s e s which a r e p r o d u c e d . 6. O n l y i n M i s s e z u l a Lake i s the r a t e o f h y b r i d i z a t i o n h i g h enough, r e l a t i v e t o the abundance o f e i t h e r p a r e n t a l s p e c i e s , to sugges t t h a t gamete l o s s through h y b r i d i z a t i o n may a f f e c t the p o p u l a t i o n s i z e o f a p a r e n t a l s p e c i e s . In the o t h e r known h y b r i d l o c a l i t i e s , b o t h p a r e n t a l s p e c i e s have h i g h l e v e l s o f abundance, and h y b r i d s c o n s t i t u t e 1.3 p e r c e n t o r l e s s o f the p a r e n t a l p o p u l a t i o n s . 7 . The p r e s e n c e o f dominance e f f e c t s i n the i n h e r i t a n c e o f some p a r e n t a l c h a r a c t e r s , combined w i t h the observed p a r t i a l f e r t i l i t y o f the h y b r i d s , the s i m i l a r i t y i n chromosome morphology, and the p r e s e n c e o f b a c k c r o s s e s i n the w i l d sugges t t h a t A c r o c h e i l u s and P t y c h o c h e i l u s a r e g e n e t i c a l l y v e r y s i m i l a r . 72 LITERATURE CITED A n d e r s o n , E . 1949. I n t r o g r e s s i v e h y b r i d i z a t i o n . J . W i l e y , New Y o r k : i x + 109. 1953. I n t r o g r e s s i v e h y b r i d i z a t i o n . B i o l . Rev. 28: 280-307. B i g e l o w , R . S . 1965. H y b r i d zones and r e p r o d u c t i v e i s o l a t i o n . E v o l u t i o n , L a n c a s t e r , P a . 19: 449-458. D a r l i n g t o n , C D . 1958. E v o l u t i o n o f g e n e t i c sys tems . O l i v e r & Boyd , E d i n b u r g h : x i i + 265. E p l i n g , C a r l 1947. G e n e t i c a s p e c t s o f n a t u r a l p o p u l a t i o n s . A c t u a l and p o t e n t i a l gene f low i n n a t u r a l p o p u l a t i o n s . Am. N a t . 81: 104-113. G i l b e r t , C a r t e r R. 1961. H y b r i d i z a t i o n v e r s u s i n t e r g r a d a t i o n : an i n q u i r y i n t o the r e l a t i o n s h i p o f two c y p r i n i d f i s h e s . C o p e i a , 1961: 181-192. Hubbs, C a r l L . 1955. H y b r i d i z a t i o n between f i s h s p e c i e s i n n a t u r e . S y s t . Z o o l . 4: 1-20. Hubbs, C a r l L . and L . C . Hubbs, 1932a. A p p a r e n t p a r t h e n o -g e n e s i s i n n a t u r e i n a form o f f i s h o f h y b r i d o r i g i n . S c i e n c e , N . Y . 76: 628-630. 1932b. E x p e r i m e n t a l v e r i f i c a t i o n o f n a t u r a l h y b r i d i z a t i o n between d i s t i n c t genera o f s u n f i s h e s . Pap. M i c h . A c a d . S c i . 15: 427-437. 1933. The i n c r e a s e d growth, predominant maleness , and a p p a r e n t i n f e r t i l i t y o f h y b r i d s u n f i s h e s . Pap . M i c h . A c a d . S c i . 17: 613-641. Hubbs, C a r l L . , L . C . Hubbs and Raymond E . Johnson, 1943. H y b r i d i z a t i o n i n n a t u r e between s p e c i e s o f s u c k e r s . C o n t r . L a b . V e r t e b r . B i o l . - U n i v . M i c h . 22: 1-76. Hubbs, C a r l L . and K . F . L a g l e r , 1947. F i s h e s o f the G r e a t Lakes R e g i o n . B u l l . C r a n b . I n s t . S c i . 26: 1-186. Hubbs, C a r l L . and L . P . S c h u l t z , 1931. The s c i e n t i f i c name o f the Columbia R i v e r chub. O c c . Pap . Mus. Z o o l . U n i v . M i c h . 232: 1-7. 73 Hubbs, C a r l L . , Boyd W. Walker and Raymond E . Johnson, 1943. H y b r i d i z a t i o n i n n a t u r e between s p e c i e s o f Amer ican c y p r i n o d o n t f i s h e s . C o n t r . L a b . V e r t e b r . B i o l . U n i v . M i c h . 23: 1-21. Hubbs, C l a r k , 1957. D u r a t i o n o f sperm v i a b i l i t y as a f a c t o r i n f r e q u e n c y o f f i s h - h y b r i d i z a t i o n . T e x . J . S c i . 9: 472-474. 1958. F e r t i l i t y o f F- j_hybr ids between the p e r c i d f i s h e s , Etheostoma s p e c t a b i l e and E . l e p i d u m . C o p e i a , 1958: 57-59. 1959. L a b o r a t o r y h y b r i d combinat ions among e theostoraat ine f i s h e s . T e x . J . S c i . 11: 49-56 . 1960. D u r a t i o n o f sperm f u n c t i o n i n the p e r c i d f i s h e s Etheostoma l e p i d u m and _E. s p e c t a b i l e , a s s o c i a t e d w i t h sympatry o f the p a r e n t a l p o p u l a t i o n s . C o p e i a , 1960: 1-8. 1961. D i f f e r e n c e s between the gamete c o m p a t i b i l i t y o f Etheostoma l ep idum sperm and eggs o f r e l a t e d p e r c i d f i s h e s from a l l o p a t r i c and s y m p a t r i c p o p u l a t i o n s . Tex . J . S c i . 13: 427-433. Mayr, E r n s t , 1963. 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DeLacey, 1935. F i s h e s o f the Amer ican N o r t h w e s t . A c a t a l o g u e o f the f i s h e s o f Washington and Oregon, w i t h d i s t r i b u t i o n a l r e c o r d s and a b i b l i o g r a p h y . J . P a n - P a c i f . Res . I n s t n . 1 0 : 366-380. 74 1936. Addenda to P a r t I . J . P a n - P a c i f . Res . I n s t n . 11: 211-290. S c h u l t z , L . P . and M i l n e r B . S c h a e f e r , 1936. D e s c r i p t i o n s o f new i n t e r g e n e r i c h y b r i d s between c e r t a i n c y p r i n i d f i s h e s o f the n o r t h w e s t e r n U n i t e d S t a t e s . P r o c . B i o l . S o c . Wash. 49: 1-10. S i n n o t t , Edmund W., L . C . Dunn and T . Dobzhansky, 1958. P r i n c i p l e s Of g e n e t i c s . M c G r a w - H i l l , New Y o r k : x i v + 459. Snyder , John O t t e r b e i n , 1905. C r i t i c a l no tes on M y l o c h e i l u s  l a t e r a l i s and L e u c i s c u s c a u r i n u s . R e p t . U . S . Comranr. F i s h . 1904: 341-342. 1908. F i s h e s o f the c o a s t a l s treams o f Oregon and N o r t h e r n C a l i f o r n i a . B u l l . B u r . F i s h . Wash. 27, 1907: 153-159. Townsley , P . M . , H . G . W r i g h t and M . A . S c o t t , 1963. M a r i n e f i s h t i s s u e c u l t u r e . J . F i s h . Res . B d . C a n . 20: 679-684. W e i s e l , George F . , 1954. A r e d i s c o v e r e d c y p r i n i d h y b r i d from Western Montana, M y l o c h e i l u s caur inum x R i c h a r d s o n i u s  b a l t e a t u s b a l t e a t u s . C o p e i a , 1954: 278-282. 1955a. The o s t e o l o g y o f M y l o c h e i l u s caur inum x P t y c h o c h e i l u s oregonense , a c y p r i n i d h y b r i d , compared w i t h i t s p a r e n t a l s p e c i e s . J . Morph . 96: 333-358. 1955b. Three new i n t e r g e n e r i c h y b r i d s o f c y p r i n i d f i s h e s from Western Montana, Am. M i d i . N a t . 53: 396-411. W o l f e , K . and C . E . Dunbar, 1958. An e x p l a n a t i o n o f the p r i n c i p l e s and methods o f t i s s u e c u l t u r e . P r o g v e . F i s h C u l t . 20: 3 - 7 . W o l f e , K . , M . C . Quimby, E . A . P y l e and R . P . D e x t e r , 1960. P r e p a r a t i o n o f monolayer c e l l c u l t u r e s from t i s s u e s o f some lower v e r t e b r a t e s . S c i e n c e N . Y . 132: 1890-1891. 

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