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The role of allelic variation in the management of fishes Gauldie, Robert William 1983

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THE ROLE OF ALLELIC VARIATION IN THE MANAGEMENT OF FISHES by ROBERT WILLIAM GAULDIE B.Sc . (Hons . l ) , The University of Otago, 1972 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Zoology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March 1982 (£) Robert William Gauldie, 1983 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make 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 study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or 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 gain s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o f 35^ The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date t5 3E-6 (3/81) As k i n g f i s h e r s catch f i r e , dragonflies draw flame; As tumbled over rim i n roundy wells Stones r i n g ; l i k e each tucked s t r i n g t e l l s , each hung b e l l s Bow sung finds tongue to f l i n g out broad i t s name-Each mortal thing does one thing and the same; Deals out that being indoors each one dwells; Selves-goes i t s e l f ; myself i t speaks and s p e l l s , Crying what I do i s me: f o r that I came. Gerard Manley Hopkins THESIS ABSTRACT (Research Supervisor: Judith H. Myers.) i i i , The t h e s i s consists of four papers that deal with the development of a b i o l o g i c a l l y reasonable r o l e f o r pr o t e i n a l l e l e s i n f i s h e r i e s management. Paper 1: The need f o r an understanding of the b i o l o g i c a l properties of p r o t e i n a l l e l e s i s established by arguments that the neutral theory of a l l e l i c c v a r i a t i o n i s an e f f e c t i v e n u l l hypothesis to the Fisher/Haldane/Wright d e f i n i t i o n of selec-t i o n , but not a legitimate explanation f o r a l l e l i c v a r i a t i o n i n i t s own r i g h t . The l i t e r a t u r e demonstrating physio-l o g i c a l d ifferences between a l l e l e s i n f i s h i s reviewed. General biochemical and various e c o l o g i c a l arguments are put forward to support the empirical evidence f o r s i g n i f i c a n t p h y s i o l o g i c a l differences between a l l e l e s . The three main empirical arguments of neutral t h e o r i s t s , the segregational load argument, the molecular clock analogy, and the c o r r e l a t i o n between polymorphism and the molecular weight of proteins, have had a great influence on p r a c t i c a l a n a l y s i s and experimental design of f i s h e r i e s management experiments i n a l l e l e frequency. The v a l i d i t y of these empirical arguments i s questioned. S t a t i s t i c a l problems associated with the n e u t r a l i s t usage of a l l e l i c v a r i a t i o n i n f i s h e r i e s management are b r i e f l y reviewed. Paper 2: T r a n s f e r r i n a l l e l e s are widely used i n f i s h e r i e s management as neutral markers. The b i o l o g i c a l r o l e of t r a n s f e r r i n a l l e l e s i s reviewed and the e f f e c t s of the b i o l o g i c a l prop-e r t i e s and the s t a t i s t i c a l use of t r a n s f e r r i n v a r i a n t s are discussed. i v Paper 3: The b i o l o g i c a l r o l e of a l l e l e s requires some r e l a t i o n s h i p between a l l e l i c variants and the parameters of f i s h e r i e s management models before a l l e l i c v a r i a t i o n can be assigned a p r a c t i c a l r o l e i n f i s h e r i e s management. Such a r e l a t i o n -ship i s demonstrated between a l l e l i c genotypes at the phosphoglucomutase locus i n l i v e r and growth i n the f i s h Cheilodactylus macropterus. Arguments are also presented for a s i g n i f i c a n t change i n a l l e l e frequency i n t h i s species due to f i s h i n g pressure. Paper 4: A b i o l o g i c a l r o l e of a l l e l e s i n f i s h e r i e s management that i s based on p h y s i o l o g i c a l differences between a l l e l e s may require an extensive understanding of the physiology and biochemistry of the f i s h before the a l l e l i c v a r i a t i o n can be put to any p r a c t i c a l use. Experimental data are presented to show that the metals Fe, Zn, P, Sr, and Na i n the f i s h o t o l i t h r e f l e c t the ambient temperature. Thus an i n d i v i -dual temperature l i f e - h i s t o r y of a f i s h may be found i n the o t o l i t h providing one, of the important experimental para-meters necessary to understanding the biochemical and physi i o l o g i c a l r o l e of a l l e l i c v a r i a n t s . The thesis concludes with a b r i e f and general overview of f i s h e r i e s manage-ment models and the reasons why the b i o l o g i c a l r o l e of a l l e l i c v a r i a t i o n i s pertinent to these models. V ACKNOWLEDGEMENTS,;. During the development of my th e s i s I was fortunate i n being able to e l i c i t much advice and c r i t i c i s m from many f a c u l t y members of the Univerr-s i t y of B r i t i s h Columbia, e s p e c i a l l y Dr Conrad Wehrhahn, Dr N.J. Wilimovsky, Mr N e i l G i l b e r t and Dr P.W. Hochachka. Members of my research committee, Dr Judith H. Myers, Dr Don McPhail, Dr Peter Larki'n, Dr T. Mulligan ( P a c i f i c B i o l o g i c a l Station) and Dr H. Tsuyuki (Canada F i s h e r i e s and Oceans) provided me with much sound advice. The experimental work on f i s h o t o l i t h s was made possible by f i n a n c i a l and material assistance from Canada F i s h e r i e s and Oceans. I received continuous assistance i n the course of my stay at the Uni v e r s i t y of B r i t i s h Columbia from many known and unknown members of the te c h n i c a l s t a f f of the u n i v e r s i t y . I am greatly indebted to a l l these people, but p a r t i c u l a r l y to Mrs Anne Nelson of the Ecology Library and Dr David Z i t t i n of the IARE computing centre. I was supported i n my course of study by the New Zealand Research Advisory Council. I thank my wife f o r her patience and support i n the development of t h i s t h e s i s . v i TABLE OF CONTENTS Page Abstract i i i Acknowledgements v L i s t of tables v i i L i s t of figures v i i i GENERAL INTRODUCTION 1 Chapter 1: The r o l e of a l l e l i c v a r i a t i o n i n the management of f i s h e s . 1. The b i o l o g i c a l properties of a l l e l e s . 3 Chapter 2: The r o l e of a l l e l i c v a r i a t i o n i n the management of f i s h e s . I I . Limits to the use of variants at the t r a n s f e r r i n locus. 58 Chapter 3: The r o l e of a l l e l i c v a r i a t i o n i n the management of f i s h e s . III.. D i f f e r e n t growth curve parameters associated with a l l e l i c phenotypes at the phosphoglucomutase locus i n the t a r a k i h i (Cheilodactylus macropterus; Cheilodactylidae: T e l e o s t e i ) . 96 Chapter 4: Ion content r e l a t i o n s h i p s of Chinook salmon o t o l i t h s : Progress towards t r a n s l a t i n g the e c o l o g i c a l record of the o t o l i t h . 117 CONCLUSIONS 150 References f o r paper 1 38 References f o r paper 2 80 References f o r paper 3 114 References f o r paper 4 146 References of Conclusions 156 v i i TABLES Page Heterozygosity of GPI 14 Species of Scombridae scored against average a l l e l e s per locus 21 Time dependent v a r i a t i o n i n t r a n s f e r r i n a l l e l e frequency 68 Asymptotic length and growth c o e f f i c i e n t s f o r PGM genotypes i n Cheilodactylus macropterus 101 Temperature treatment group ion l e v e l s 127 Ion composition of o t o l i t h s 137 F i s h weight and o t o l i t h weight 140 O t o l i t h ion concentration interdependence 141 Regression of genotype frequency and Fe content of t a r a k i h i o t o l i t h s 142 v i i i FIGURES Page Average number of a l l e l e s per locus within the family Scombridae p l o t t e d against molecular weight 19 Frequency of heterozygous l o c i i n humans 29 Sex r a t i o of the t a r a k i h i 111 Range of mixed a r a g o n i t i c / v a t e r i t i c o t o l i t h s 122 O t o l i t h metal ion content at d i f f e r e n t temperatures 132-136 1 INTRODUCTION Proteins coded at a single locus frequently vary i n t h e i r p h y s i c a l properties because of differences i n t h e i r amino acid composition or sequence. While these d i f f e r e n t forms of the same pro t e i n r e t a i n s i m i l a r biochemical functions, they frequently d i f f e r i n t h e i r electrophoretic mobi li ty and are distinguishable as a l l e l e s at a p a r t i c u l a r locus. The frequency d i s t r i b u t i o n of a l l e l e s i n geographically separated samples i s widely used i n f i s h e r i e s management to define g e n e t i c a l l y i s o l a t e d stocks of f i s h . This usage i s dependent on the assumption that the electrophoretic. v a r i a t i o n which allows the i d e n t i f i c a t i o n of a l l e l e s occurs without a s i g n i f i c a n t change i n pro t e i n function. My objective i s twofold: f i r s t to show that t h i s assumption i s i:v- ";'. i n v a l i d , and second to show that the information that i s discarded by making t h i s assumption i s of greater value to f i s h e r i e s management science than the simple d e f i n i t i o n of g e n e t i c a l l y i s o l a t e d stocks. I argue that the experimental evidence, f o r differences i n function between electrophoretic a l l e l e s i s inconsistent with the assumption of p h y s i o l o g i c a l i d e n t i t y of a l l e l e s . Therefore, not only i s the use of a l l e l e s as a s t a t i s t i c a l t o o l i n stock recognition undermined by the variance that t h e i r p h y s i o l o g i q a l differences tend to generate, but also the s t a t i s t i c a l approach discards much valuable information. A p a r t i c u l a r argument against the assumption of p h y s i o l o g i c a l i d e n t i t y of a l l e l e s w i l l be made from the information a v a i l a b l e f o r the t r a n s f e r r i n locus. Further evidence f o r the p h y s i o l o g i c a l differences among a l l e l e s i s given by the asso c i a t i o n between growth and a l l e l i c phenotype i n the f i s h Gheilodactylus macropterus. Both these arguments have important implications f o r f i s h e r i e s management. 2 A c r i t i c a l p r e r e q u i s i t e for the use of p h y s i o l o g i c a l differences between a l l e l e s by f i s h e r i e s management i s a knowledge of the l i f e h i s t o r y of i n d i v i d u a l f i s h e s i n order to e s t a b l i s h when and where the p h y s i o l o g i c a l differences between a l l e l e s are important. I present experimental evidence to show that the metal ion content of the o t o l i t h s of the Chinook salmon, Oncorhynchus tshawytscha, changes with ambient temperature. Considerable t e c h n i c a l d i f f i c u l t i e s l i e i n the way of using t h i s metal ion information contained i n the o t o l i t h s , but my experience indicates the p o t e n t i a l f o r e s t a b l i s h i n g the i n d i v i d u a l temperature l i f e h i s t o r y of f i s h e s . My t h e s i s draws together information from many d i s c i p l i n e s . Rather than write a very large and unwieldly t h e s i s that integrates biochemistry, genetics and f i s h e r i e s management, I have instead resorted to a format that allows me to express d i f f e r e n t , but r e l a t e d , points. In t h i s way I hope to present to f i s h e r i e s managers new views of an o l d problem. Four papers presented i n the body of the t h e s i s are followed by a conclusion i n which the material contained i n these four papers i s drawn together to s a t i s f y the objective of my t h e s i s . The growth curve c a l c u l a -tions i n the t h i r d paper were made by W.J. Gazey. J . Davidson and Janet Pel were the technicians who c a r r i e d out the elemental analysis for the o t o l i t h paper; t h e i r co-authorship i s a courtesy which they deserve fo r t h e i r considerable t e c h n i c a l s k i l l s . The four, papers c o n s t i t u t i n g the body of my thesis w i l l be submitted to the Transacticrsof the American F i s h e r i e s Society for p u b l i c a t i o n . 3 THE ROLE OF ALLELIC VARIATION IN THE MANAGEMENT OF FISHES 1. The b i o l o g i c a l properties of a l l e l e s . * R.W. GAULDIE * F i s h e r i e s Research D i v i s i o n P 0 Box 297 Wellington, New Zealand New Zealand Research Advisory Council Fellow at The I n s t i t u t e of Animal Resource Ecology The Uni v e r s i t y of B r i t i s h Columbia 4 ABSTRACT The need f o r an understanding of the b i o l o g i c a l properties of pr o t e i n a l l e l e s i s established by arguments that the neutral theory of a l l e l i c v a r i a t i o n i s an e f f e c t i v e n u l l hypothesis t c the Fisher/Haldane/Wright d e f i n i t i o n of s e l e c t i o n , but not a legitimate explanation f o r a l l e l i c v a r i a t i o n i n i t s own r i g h t . The l i t e r a t u r e on p h y s i o l o g i c a l differences between, a l l e l e s i n f i s h i s reviewed. General biochemical and various e c o l o g i c a l arguments are put forward to support the empirical evidence f o r si g n i f i c a n t , p h y s i o l o g i c a l differences between a l l e l e s . The. three main empirical arguments of neutral t h e o r i s t s , the segrega-t i o n a l load argument, the molecular clock analogy, and the c o r r e l a t i o n between polymorphism and the molecular weight, of proteins, have had a great influence on p r a c t i c a l analysis and experimental design of f i s h e r i e s management experiments i n a l l e l e frequency. The v a l i d i t y of these empirical arguments i s questioned. S t a t i s t i c a l problems associated with the n e u t r a l i s t usage of a l l e l i c v a r i a t i o n i n f i s h e r i e s management are b r i e f l y reviewed. 5 INTRODUCTION The use of p r o t e i n a l l e l e s * i n f i s h e r i e s management depends on the properties of the a l l e l e s themselves. Two a l t e r n a t i v e properties of a l l e l e s have been proposed. F i r s t , that a l l e l e s are neutral markers r e s u l t i n g from changes i n the amino a c i d composition of proteins that a f f e c t the electrophoretic mobility of proteins without changing the p h y s i o l o g i c a l properties of the proteins. Second, that the changes i n amino a c i d composition leading to the- differences i n electrophoretic m ob il it y that d i s t i n g u i s h a l l e l e s should also r e s u l t i n biochemical differences that lead to physiological, differences between a l l e l e s . In a f i s h e r i e s management context these a l t e r n a t i v e properties lead to a l t e r n a t i v e p o t e n t i a l uses of p r o t e i n a l l e l e s . I f a l l e l e s are neutral markers of the genome they can be used to i d e n t i f y separate breeding populations within a species. This usage a r i s e s from the d e f i n i t i o n of separate breeding populations as being randomly mating groups that do not mate with other groups i n the same species. I f a l l e l e frequency differences between groups are greater than i s expected by chance, then those groups must be g e n e t i c a l l y i s o l a t e d from each other since the random mating process must lead to genetic homogeneity. These g e n e t i c a l l y i s o l a t e d populations are r e f e r r e d to as 'stocks'. The existence of 'stocks' i s tested by taking samples over the species range. The sampling process can follow a number of s t a t i s t i c a l models (Gorman and Renzi, 1979; Allendorf and Utter, 1980) with d i f f e r e n t sample s i z e s , but b a s i c a l l y a l l these models measure the o v e r a l l v a r i a t i o n a l l e l e * A l l e l e s : a l l e l e s are defined i n t h i s context as a l t e r n a t i v e proteins coded by d i f f e r e n t DNA sequences that comprise synonymous genes occurring at the same locus. This i s the common usage i n the f i s h l i t e r a t u r e (Allendorf and Utter, 1980) although ' a l l e l e ' properly r e f e r s to the gene coding f o r the p r o t e i n . 6 frequency at one or many l o c i i n terms of some s t a t i s t i c a l i n t e r r e l a t i o n -ship between frequencies. S t a t i s t i c s ranging from simple chi-square values to mul t i v a r i a t e s t a t i s t i c s can then be used to t e s t whether the-diffe r e n c e s i n a l l e l e frequencies between geographical areas are greater than expected by chance. Examples of 'stocks' defined i n t h i s way are to be found i n Allendorf and Utter (1980); other examples are given by Jamieson and Turner (1976) and by Grant and Utter (1980). The question of genetic homogeneity of 1 stocks 1 i s fundamental to f i s h e r i e s management strategy. Genetic homogeneity should not be confused with genetic homo-zygosity. Genetic homogeneity a r i s e s from random mating. The consequ-ences of genetic heterogeneity within a 'stock' are serious. For example, Ricker (1973) discussed the e f f e c t s of modelling mixed species as i f they were a sing l e species. In such circumstances catch l e v e l s based on the mean growth rate of the species mixture w i l l tend to o v e r f i s h the slowest growing species and drive the o v e r a l l growth rate towards that of the fa s t e s t growing species. I f p h y s i o l o g i c a l differences between a l l e l e s lead to d i f f e r e n t i n d i v i d u a l growth rates, then Ricker's (1973) argument can be extended to cover a l l e l i c a l l y heterogeneous groups of f i s h . In such a case catches based on mean growth rate would lead to the loss of the slower growing p h y s i o l o g i c a l variants and drive the population towards a monoculture of the f a s t e s t growing p h y s i o l o g i c a l v a r i a n t s . This s i t u a t i o n may or may not be a desirable development i n the o v e r a l l s t a b i l i t y of a f i s h e r y . S i m i l a r l y , i f disease resistance, fecundity, and other p h y s i o l o g i c a l t r a i t s depend on the i n d i v i d u a l ' s a l l e l i c genotype, then the a l l e l e frequency d i s t r i b u t i o n of populations within a species can no longer be assumed to represent a g e n e t i c a l l y i s o l a t e d 'stock', since 'genetically 7 i s o l a t e d ' requires that there be l i t t l e or no migration between stocks so that differences i n a l l e l e frequency are due to i s o l a t i o n . Even a minor amount of migration between 'neutral' stocks leads to genetic homogeneity. A kind of stock would s t i l l e x i s t i f there were p h y s i o l o g i c a l differences between a l l e l i c genotypes, and such stocks could s t i l l be s t a t i s t i c a l l y d istinguished from each other, but the meaning of such a 'stock' from a f i s h e r i e s management viewpoint would be quite d i f f e r e n t from the meaning of 'stocks' defined by .'neutral' a l l e l i c v a r i a t i o n since these 'stocks' would be g e n e t i c a l l y heterogeneous. Thus two quite d i f f e r e n t f i s h e r i e s management problems are accommoer dated by regarding a l l e l i c v a r i a t i o n as being on the one hand a 'neutral' marker and on the other as having p a r t i c u l a r p h y s i o l o g i c a l properties. Most published studies of a l l e l i c v a r i a t i o n of f i s h e s favour the use of a l l e l i c v ariants as simple population characters whose frequencies can be used s t a t i s t i c a l l y to assign 'stocks' within a species. This i s not to say that most authors claim that a l l e l i c v a r i a t i o n i s always neutral, although many do. Nonetheless the f i n a l outcome of nearly a l l a l l e l i c studies i s a statement about the s t a t i s t i c a l s i g n i f i c a n c e of differences i n frequencies between samples, which implies that a l l e l e frequencies are constant over time. There i s no reason why s t a t i s t i c a l analyses aimed at recognising patterns of d i s t r i b u t i o n l i k e 'stocks' cannot be c a r r i e d out when there are p h y s i o l o g i c a l differences between a l l e l e s . But such studies require much more complex sampling procedures to overcome the s t a t i o n a r i t y assumption discussed below. How would one show that the a l l e l i c variants were not 'neutral' markers but p h y s i o l o g i c a l l y d i f f e r e n t proteins? 8 In "The Genetic Basis of Evolutionary Change" Lewontin (1974) sought to e s t a b l i s h whether the neutral theory models could account f o r the observ-ed v a r i a t i o n i n a l l e l i c frequencies as well as s e l e c t i o n theory models. He concluded that they could. This i s not the question that we are addressing. I regard Lewontin's (1974) study.as a demonstration.that s e l e c t i o n models based on the c l a s s i c Haldane/Fisher/Wright d e f i n i t i o n of s e l e c t i o n * do not explain the d i s t r i b u -t i o n of a l l e l i c v a r i a t i o n any better than the n u l l hypothesis provided by the neutral theory. Lewontin (1974) d i d not show that s e l e c t i o n was not responsible f o r observed patterns of a l l e l i c v a r i a t i o n . He showed that a p a r t i c u l a r model of s e l e c t i o n could not account f o r '..the J observed patterns of a l l e l i c v a r i a t i o n any better than a n u l l hypothesis based on neutral v a r i a t i o n . A model of s e l e c t i o n that i s consistent with the data i s not a necessary p r e r e q u i s i t e to the question of whether or not there are physio-l o g i c a l d ifferences between a l l e l e s . Nor i s such a model a necessary p r e r e q u i s i t e to the a p p l i c a t i o n of the knowledge of p h y s i o l o g i c a l d i f f e r -ences between a l l e l e s to f i s h e r i e s management problems. I t i s the reverse that i s necessary. T h e o r e t i c a l models cannot be made without understand-ing the empirical basis f o r p h y s i o l o g i c a l differences between a l l e l e s . Therefore, the important question i s whether there i s any experimental evidence of p h y s i o l o g i c a l . d i f f e r e n c e s associated with electrophoretic v a r i a t i o n i n f i s h e s and other aquatic organisms. I review here a number of studies which I f e e l provide an a f f i r m a t i v e answer to t h i s question. * Haldane, Fisher and Wright d i d not have convergent views on s e l e c t i o n , but they d i d use the same formal d e f i n i t i o n of s e l e c t i o n . 9. DEMONSTRABLE PHYSIOLOGICAL DIFFERENCES BETWEEN ALLELES The most c l e a r l y demonstrated case of p h y s i o l o g i c a l v a r i a t i o n among in d i v i d u a l s with d i f f e r e n t enzyme a l l e l e s i s the p h y s i o l o g i c a l r o l e of leucine amino peptidase in.Mytilus e d u l i s and the subsequent geographical d i s t r i b u t i o n of a l l e l e s at that locus (Koehn, 1978; Koehn et a l . , 1980; Koehn and Immerman, 1981; Koehn and Siebenaller, 1981) . Koehn shows that Mytilus e d u l i s has short-term regulatory physiologies concerned with leucine amino peptidase a c t i v i t y ; i n which p h y s i o l o g i c a l l y d i s t i n c t variants of the enzyme are associated with the rapid establishment of regulatory responses i n d i f f e r e n t environments. For the rainbow trout, Salmo g a i r d n e r i , numerous studies from various .laboratories show biochemical, behavioural, and p h y s i o l o g i c a l differences between i n d i v i d u a l s with d i f f e r e n t l a c t a t e dehydrogenase a l l e l e s (Northcote, W i l l i s c r o f t and Tsuyuki, 1970; Tsuyuki and W i l l i s c r o f t , 1973; Huzyk and Tsuyuki, 1974; Bailey, Tsuyuki and Wilson, 1976; Tsuyuki and W i l l i s c r o f t , 1977; Kao and Farley, 1978; K l a r , Stalnaker and Farley, 1979a,b; Redding and Schreck, 1979; Northcote,c.l9.81; Northcote and Kelso, 1981) . Similar studies i n other f i s h e s show a correspondence between a l l e l e function dependency on the environment and the geographical d i s t r i b u t i o n of those a l l e l e s . These fi s h e s include the k i l l i f i s h , Fundulus h e t e r o c l i t u s (Mitton and Koehn, 1975; Powers and Powers, 1975; Place and Powers, 1978; Powers and Place, 1978; .Powers, Greaney and"place, 1979), the blenny, Anoplarchus purpurescens (Johnson, 1971a,b) and the shiners, Notropis  stramineus (Koehn, 1970; Koehn, Perez and M e r r i t t , 1971), Notropis  l u t r e n s i s (Richmond.and Zimmerman, 1978), and the fathead minnow Pimephales promelas (Merritt, 1972). For the p r o t e i n t r a n s f e r r i n , recent studies have shown that d i f f e r e n t a l l e l e s confer d i f f e r e n t resistance to i n f e c t i o n by various pathogens (Pratschner, 1977; Suzomoto, Schreck and Mclntyre, 1979). T r a n s f e r r i n s have been known as b a c t e r i o c i d a l agents f o r a long time and a l l e l i c v a r i a -t i o n of t r a n s f e r r i n i n man, bi r d s and farm animals i s widely accepted as being due to differences i n disease resistance and differences i n some "general condition" f a c t o r i n animal, physiology. This t o p i c , and i t s consequences f o r f i s h e r i e s stock studies where t r a n s f e r r i n s are widely used, i s the subject of the next paper i n t h i s s e r i e s (Gauldie, 1982) and w i l l not be pursued i n d e t a i l here. _The studies quoted above are convincing i n t h e i r own r i g h t and i n good accord with what i s known about the p h y s i o l o g i c a l e f f e c t s of the various enzymes involved (e.g., f o r l a c t a t e dehydrogenase, Hochachka and Lewis, 1971; Malan, Wilson, and Reeves, 1976; Yancey and Somero, 1978; Knox:, Walton and Cowey, 1980). CLINAL DISTRIBUTION OF ALLELES Many studies show c l e a r geographical c l i n e s i n a l l e l e frequencies or heter-oz y g o s i t i e s . C l i n e s are consistent with the neutral theory, but i n many studies a p a r t i c u l a r environmental, v a r i a b l e i s i d e n t i f i e d and cor r e l a t e d with the v a r i a t i o n i n allele.frequency which suggests that they are main-tained by s e l e c t i o n (Fujino and Kang, 1968; Lush, 1969; Koehn, Turano and Mitton, 1971; Pantelouris et a l . , 1971; Avise and Selander, 1972; Koehn and Mitton, 1972; Frydenberg, 1973; Christiansen and Frydenberg, 1974; Hjorth and Simonsen, 1975; Mathers, 1975; Murphy, 1976; Yardley and Hubbs, 1976; Schopf, 1977; Wilkins, 1977; Buth and Burr, 1978; Koehn and Williams, 1978; Lassen and Turano, 1978; Powers and Place, 11 1978; Shami and Beardmore, 1978; Shoubridge and Legget, 1978; Avise and F e l l e y , 1979; Hadfield et a l . , 1979; Kijima and F u i j o , 1979; Kreuger and Menzel, 1979; Gosling, 1980; Gauldie and Johnstone, 1980; Anon, 1981). The behaviour of a l l e l e frequencies i n terms of the heterozygosity being c o n t r o l l e d by the v a r i a t i o n i n the environment rather than by a s e l e c t i o n generated excess of heterozygotes has been examined by a number of authors (Levins and MacArthur, 1966; G i l l e s p i e and Langley, 1974; Soule, 1976; Valentine, 1976; Minawa and B i r l e y , 1978; Powell and Wistrand, 1978; Pierce and Mitton, .1979; MacKay, 1980). Some studies have shown that there i s year-to-year v a r i a t i o n i n a l l e l e frequency i n some f i s h e s which one might expect from heterozygosities that are a function of environ-mental variance (Wilkins, 1966; Elson, 1973; Loch, 1974; Jamieson and Turner, 1976; K r i s t i a n s e n and Mclntyre, 1976; Siebenaller, 1976; Wilkins, 1977; Yoshiyasu, 1979; Grant et a l . , 1980; Gauldie and Johnston, 1980; Smith, 1980). There remain a very large number of studies i n which differences i n a l l e l e frequency between and within populations are reported but i n which no t e s t of the n e u t r a l / s e l e c t i v e status of the v a r i a t i o n i s put forward other than Hardy-Weinberg equilibrium, the heterozygosity/environment variance argument, or simply the bald assumption that a l l of the a l l e l i c v a r i a t i o n reported has no p h y s i o l o g i c a l s i g n i f i c a n c e . To r e s t r i c t the s i z e of t h i s review, I w i l l not l i s t them but simply characterise them as I have done above. A small group of studies describe populations that have stable a l l e l e frequencies e i t h e r over distance or time (Smith and Jamieson, 1977; Grant and Utter, 1980; S i d e l l et a l . , 1980) or are v i r t u a l l y f i x e d at many l o c i (Smithyand\.McK6y,/1978;, Felley. and Avise, 1980) . There may be many more i n v a r i a n t species that have not been reported because of the tendency to equate high l e v e l s of v a r i a t i o n with b i o l o g i c a l importance. This section may be summarized as follows. (1) There i s convincing evidence f o r p h y s i o l o g i c a l differences between some a l l e l e s i n some f i s h and molluscs, and that the geographical d i s t r i b u -t i o n of these a l l e l e s i s c o n t r o l l e d by the p h y s i o l o g i c a l differences between a l l e l e s . (2) There are d i r e c t and t h e o r e t i c a l biochemical reasons to indicate that the charge difference between a l l e l e s i s i t s e l f s u f f i c i e n t basis to strongly suspect that there are p h y s i o l o g i c a l differences between a l l e l e s . (3) There are a number of studies reported i n the l i t e r a t u r e which report c l i n e s i n a l l e l e frequencies which may be due to p h y s i o l o g i c a l d i f f e r e n c e s between a l l e l e s . Thus the weight of experimental evidence supports an i n t e r p r e t a t i o n of genetic v a r i a t i o n i n terms of p h y s i o l o g i c a l differences between a l l e l e s . S i m ilar arguments hold f o r a l l e l i c v a r i a t i o n i n some organisms other than f i s h and molluscs (Leigh-Brown, 1977; Bijlsma, 1978; Kamping and van Delden, 1978; Bijlsma and van der Meulen-Bruijns, 1979; Danforth and Beardmore, 1979; Fucci et a l . , 1979; Kokima and Tbbari.,, 1979; Lee and Pegoraro, 1979; Mason, 1979; Narise, 1979; Barker and East, 1980; Hoorn and Scharloo, 1980). Not a l l neutral models of biochemical function are t h e o r e t i c a l . Three important empirical neutral arguments are: the segregational load argument, the dependence of a l l e l i c v a r i a t i o n on molecular weight, and the molecular clock analogy. These empirical arguments have had a great influence.on the actual analysis of a l l e l e frequency data i n f i s h e r i e s 13 management and so require further comment. THE CORRELATION BETWEEN THE NUMBER OF ALLELIC VARIANTS AND THE MOLECULAR  WEIGHT OF PROTEINS I t i s reasonable to suppose that i f a l l e l i c v a r i a n t s of proteins are jus t chance changes i n amino acids, then the number of a l l e l e s should increase with increasing molecular weight. This hypothesis i s easy to t e s t since molecular weights of a given enzyme t y p i c a l l y do not vary greatly between widely d i f f e r e n t animals. For example the molecular weights of various l a c t a t e dehydrogenases from salmon to humans vary by only a few percent (Boyer, 1975)\ However, when the v a r i a t i o n i n the number of a l l e l e s found i n a p a r t i c u l a r protein (or proteins of s i m i l a r s i z e .(Ward, 1978)) i s examined i t proves.to be very great indeed. Table 1, from Gauldie and Smith (M.S.),Mists the frequency of a l l e l e s at the glucose phosphate isomerase locus. The number of a l l e l e s per locus shown by glucose phosph-ate isomerase i s by no means unusually large. The t r a n s f e r r i n locus has-seventeen a l l e l e s i n the cod Gadus morhua (Jamieson and Turner, 1976). Sheep and c a t t l e have almost as many transf errih'-.alleles (see Gauldie, 1982) although t r a n s f e r r i n v a r i a t i o n i n other vertebrates l i k e b i r d s (Frelinger, 1972) and A t l a n t i c salmon (Payne, C h i l d and Forrest, 1971) i s l i m i t e d to two a l l e l e s . In the face of t h i s kind of within-locus v a r i a t i o n , one would expect to f i n d low c o r r e l a t i o n s between the number of a l l e l e s and the molecular weight of proteins. Thus i t i s not s u r p r i s i n g then that the r 2 values f o r the c o r r e l a t i o n between molecular weight and heterozygosity are low, .003 to .155 (Ne'i eet sal , . , , 1978). The average c o r r e l a t i o n of heter-ozygosity with molecular weight reported over species by Nei.et a l . (1978) i s .079 to .522, but t h i s highest value,.522 f o r Drosophila, i s claimed to 14 Table 1. This table i s i n part reproduced from Gauldie and Smith (M.S.), and includes data from a number of species of f i s h . Heterozy-gosity (HZ) i s c a l c u l a t e d by (2pg), that i s , a f r a c t i o n of 1.00 GLUCOSEPHOSPHATE ISOMERASE ALLELE FREQUENCIES N +3 +2 +1 0 -1 -2 -3 Hz 202 .007 vO50 .921 .0220 .149 30 1.000 0 741 .005 .303 .529 .1680 .600 30 .017 .983 .033 295 .003 .322 .576 .0980 .007 .550 260 .200 .763 .0370 .380 1475 .004 .976 .0200 .047 395 .002 .491 .4390 .068 .560 976 .006 .166 .777 .0470 .003 .370 627 .070 .800 .1300 .340 172 .596 .4040 .480 319 1.000 0 3760 . .106 .286 .608 .0008 .540 30 1.000 0 229 .009 .229 .162 .450 .1440 .006 .698 226 .009 .529 .4400 .020 .002 .530 164 .150 .700 .1500 0.465 64 .805 .1790 .320 136 1.000 0 257 .010 .001 .020 .930 .0300 .007 .130 28 .810 .1900 .310 52 .970 .0300 .060 22 1.000 0 14 .980 .0200 .040 28 .750 .2500 .375 28 .930 .0700 .130 60 1.000 0 25 1.000 0 25 1.000 0 25 1.000 0 25 1.000 0 25 1.000 0 GLUCOSEPHOSPHATE. ISOMERASE ALLELE FREQUENCIES (Continued) N +3 +2 +1 0 -1 -2 -3. Hz 25-200 25-200 25-200 25 very large 100 100 23 .020 11 .090 0.000 34 .180 48 .040 .040 68 .250 32 14 149 77 1.000 0 1.000 0 1.000 0 1.000 0 1.000 0 1.000 0 .976 .0240 .050 .870 .1100 .230 .550 0.0000 .360 .559 .820 .295 .920 .150 .750 .375 1.000 0 1.000 0 .997 .0030 .006 .870 .1300 .230 17 be an over-estimate f o r which the true value should be .11 (Leigh-Brown and Langley, 1979). Leigh-Brown and Langley (1979) also point out that such a low c o r r e l a t i o n i s just as r e a d i l y predicted by other, including s e l e c t i o n -i s t , hypotheses. Nei e t a a l . (1978) t r i e d to explain these observations, including the observation by Hopkinson, Edwards and Harris (1976) that there was no s i g n i f i c a n t d i f f e r e n c e i n molecular weight between polymorphic and mono-morphic l o c i i n human beings, by arguing that a strong c o r r e l a t i o n i s masked by the stochastic l o s s of a l l e l e s ( d r i f t effects) due to differences i n e f f e c t i v e population s i z e . Nei et a l . (1978) support t h i s argument i n a somewhat round-about fashion. E f f e c t i v e population size cannot be measured experimentally; i t has to be estimated from the d i s t r i b u t i o n of a l l e l e frequencies. Since heterozygosity i s an i n d i r e c t measure of a l l e l e frequency, i t must be highly c o r r e l a t e d with estimated e f f e c t i v e population s i z e . An estimate of the stochastic l o s s measured from the e f f e c t i v e population si z e v i a heterozygosity cannot be applied to the o r i g i n a l data set without invoking a c i r c u l a r argument. Unfortunately, Nei et a l . (1978) avoid t h i s c i r c u l a r i t y only by introducing other c i r c u l a r i t i e s . They obtain the t h e o r e t i c a l value of the proportion of a l l e l i c v a r i a t i o n that i s explained by molecular weight, " i f we assume that the mutation rate f o r a locus i s proportional to the mole-cular weight of the polypeptide produced",, which i s , of course, the very thing that the paper sets out to t e s t . I conclude that the n e u t r a l i s t explanation f o r the very low r 2 values between a l l e l i c v a r i a t i o n and molecular weight as being due to d r i f t e f f e c t s i s very weak. 18 F i g . 1 The average number of a l l e l e s per locus f o r 18 species within the family Scombridae (from Sharp and Pirages (1978)) p l o t t e d against the subunit molecular weight of the 18 enzymes examined. 20 In contrast to the neutral arguments, i t has been demonstrated that c e r t a i n p h y s i o l o g i c a l properties of proteins, p a r t i c u l a r l y s t a b i l i t y i n sol u t i o n and net a l k a l i n e or acid surface charge, are correlated with molecular weight (Momany' eti'.al. , 1976; K i l l i l e a et a l . , 1978; Dice et a l . , 1979; Rocha et a l . , 1979). Consequently, one might look f o r some c o r r e l a -t i o n between a l l e l i c v a r i a t i o n and p h y s i o l o g i c a l function, and f i n d i t i n the a l l e l i c v a r i a t i o n reported by Sharp and Pirages (1978) f o r some of the species i n the family Scombridae. They l i s t the number of electrophoretic variants at 21 l o c i (14 enzymes) f o r 21 species (see Table 2). The 21 species examined include both the highly s p e c i a l i s e d homeotherms l i k e Thunnus thynnus and unspecialised poikilotherms l i k e the s i e r r a s and bar mackerels (Scombromorus). The m o b i l i t i e s of a l l electrophoretic variants were measured r e l a t i v e to the corresponding locus i n the y e l l o w f i n tuna (Thunnus albacares). Leigh-Brown et a l . (1979), i n t h e i r examination of a l l e l i c v a r i a t i o n , went to some trouble to accurately measure molecular weight. This does not seem necessary. Sequencing data from Boyer (1975) indi c a t e that the molecular weights of beef l a c t a t e dehydrogenase and malate dehydrogenase d i f f e r from those of tuna and salmon by only a few percent. The biggest diffe r e n c e i s between beef and horseshoe crab l a c t a t e dehydrogenase, but t h i s i s s t i l l only about 5% (Boyer, 1975) . Consequently, I have used human enzyme molecular weight data from Harris and Hopkinson (1976) to t e s t the r e l a t i o n s h i p between the number of a l l e l e s and molecular weight across a l l 21 species of scombrids (Fig. 1). A p o s i t i v e association between the number of a l l e l e s and molecular weight i s not supported. The family Scombridae covers r e l a t e d genera that range from s p e c i a l i s e d homeotherms l i k e Thunnus thynnus to unspecialised poikilotherms l i k e 21 Table 2. Species within the family Scombridae are scored against t h e i r respective average number of a l l e l e s per locus. Species within a common genus are designated by a l e t t e r standing f o r the follow-ing genera from C o l l e t t e (1978): A, Scomberomorus; B, Scomber; C, Sarda; E, Euthynnus; G, subgenus Thunnus; H, subgenus Neothunnus; I, subgenus Parathunnus. The superscript numbers on the l e t t e r s i n d i c a t e the l e v e l of homeothermy described i n the text. 22 Table 2 Species 1.29 Thunnus thynnus (H) 1" Thunnus maccoyi (H) 1' 1.31 1.33 1.35 Thunnus o r i e n t a l i s (H) 1.37 Euthynnus a f f i n i s (E) 1.39 Thunnus tonggol (G) 1" 1.41 Scomberomorus concolor. (A) 2. 2 1.43 Thunnus alalunga (H) " Thunnus albacares (G) 1.45 Acanthocybium solanderi (A) 1.47 Sarda c h i l i e n s i s (C), Thunnus obesus (1) 1.49 1.51 Thunnus a t l a n t i c u s (G) 1.53 Scomberomorus c a v a l l a (A) 1.55 Scomber japonicus (B) 1.57 1.59 1.61 1.63 1.65 1.67 1.69 1.71 Katsuwonus pelamis (F) 1.73 1.75 Auxis thazard (D), Euthynnus lineatus (E) 1.77 1.79 1.81 Scomberomorus s i e r r a (A) 23 Scomber japonicus. I f the number of a l l e l e s per locus i s a function of the environment, one would expect a progressive decline i n the number of a l l e l e s per locus from the poikilothermic to the homeothermic scombrids. The average number of a l l e l e s per locus l i s t e d by Sharp and Pirages (1978) i s shown by species i n Table 2. In a general way, Table 2 does indeed show that the most s p e c i a l i s e d homeotherms have the l e a s t a l l e l i c v a r i a t i o n , but there are many f i s h e s - l i k e the poikilothermic Scomberomorus concolor that show too l i t t l e v a r i a t i o n to f i t the proposed r e l a t i o n s h i p . However, since a l l e l i c v a r i a t i o n i s presum-ably s e n s i t i v e to competition between r e l a t e d species within c e r t a i n envir-onments, a f a i r e r comparison may be between species within genera. Unfortunately, Sharp and Pirages (1978) provide data on only 18 of the 46 species i n the family so that only two sub-genera, Neothunnus and Thunnus, provide enough species (3 and 4 respectively) to t e s t the v a r i a t i o n between species within general. Within the sub-genus Thunnus, the b l u e f i n species Thunnus thynnus and Thunnus maccoyi are l e s s v a r i a b l e than t h e i r cogener Thunnus alalunga, and there i s evidence ( C o l l e t t e , 1978, plates 6 and 7) that the b l u e f i n s do have the more advanced heat exchange system. S i m i l a r l y , i n the sub-genus Neothunnus, the more advanced Thunnus tonggol has fewer variants than i t s le s s sophisticated homeothermic cogener Thunnus albacares. These speculations are s e r i o u s l y compromised by the uneven sample siz e s of Sharp and Pirage's (1978) data, but the trends that emerge from the data reported f o r the family Scombridae cast considerable doubt on any simple r e l a t i o n s h i p between a l l e l i c v a r i a t i o n and molecular weight. On the contrary, the trend i s consistent with.the view that polymorphism i s t i e d to the environment and physiology of scombrids. 24 The empirical r e l a t i o n s h i p between a l l e l i c v a r i a t i o n and the molecular weight of proteins cannot be regarded as favourable evidence f o r the neutral theory. THE "MOLECULAR CLOCK" ANALOGY This analogy r e f e r s to the c o r r e l a t i o n of the genetic distance between proteins as measured by the number of amino acid substitutions at a locus and the phylogenetic distance between proteins as measured by the taxonomic status of. the organisms from which they are drawn. "Genetic distance" r e f e r s to differences based on measurements made on parts of the genome that are supposed to.be independent of the phenotypic characters used i n the taxonomy of the organism, i n t h i s case, a l l e l i c v a r i a n t s . This i s "genetic distance" i n a l i t e r a l sense rather than the formal usage of Rogers (1972). The best known of these c o r r e l a t i o n s i s that of F i t c h and Margoliash (1967) who showed that the divergence between animals as measured by the accumulation of amino a c i d substitutions p a r a l -l e l s t h e i r phylogenetic divergence measured, by t h e i r phenotypic taxonomy. So, f o r example, the number of amino a c i d s u b s t i t u t i o n d i f f e r e n c e s between a horse and l i z a r d w i l l be much greater than that between a horse" and a rabbit since the time between divergence from a common ancestor to the present i s greater f o r the l i z a r d than the rabbit. I t i s evident from our discussion on the e f f e c t s of amino a c i d s u b s t i -tutions on enzyme functions that the differences i n enzyme function should be r e f l e c t e d i n the number of changes made i n the amino a c i d composition of the enzyme. The support that the molecular clock analogy of F i t c h and Margoliash (1967) and other s i m i l a r studies lend to the neutral theory i s e n t i r e l y dependent on the assumption that a l l e l i c v a r i a n t s are p h y s i o l o g i -c a l l y n e utral, since s e l e c t i o n would lead to exactly the same r e s u l t . Since E i t c h and Margoliash (1967) and others have only modern animals to observe, they also have to assume that the horseshoe crab, and other p r i m i t i v e organisms, are unchanged r e l i c s from the past. There i s no reason to believe that modern r e l i c s are the same species as t h e i r f o s s i l progenitors. The 'modern' horseshoe .crab may be j u s t as biochemically modern, as the 'modern' horse. Other problematic assumptions are discussed by Ewens (1979). I t i s not possible to d i s t i n g u i s h between the two hypotheses (i) that differences i n amino a c i d substitution, r e f l e c t the time of phylogenetic divergence, and ( i i ) that differences i n amino acid s u b s t i t u t i o n r e f l e c t d i f f e r e n t physiologies. Consequently systematic and taxonomic arguments based on amino acid s u b s t i t u t i o n that.are aimed at e s t a b l i s h i n g phylogenetic r e l a t i o n s h i p s require, a p r i o r i , a complete acceptance of the neutral theory. THE SEGREGATIONAL LOAD THAT IS ASSOCIATED WITH SELECTION The concept of segregational load i s the very basis of the neutral theory. I t f i r s t appears i n Kimura (1960) as a development of the genetic load concepts of Haldane (1957) and Muller (1950). The argument i s very simple: i f a l l the genes i n an organism were polymorphic, and i f that polymorphism were maintained by s e l e c t i o n , then each random mating popula-t i o n would produce a vast number of d i f f e r e n t combinations of genotypes from a l l the segregating l o c i . In order for s e l e c t i o n to restore the parental frequency d i s t r i b u t i o n from the multitude of o f f s p r i n g patterns, an enormous mortality i s required. An i n d i c a t i o n of the si z e of these 26 m o r t a l i t i e s is. i l l u s t r a t e d by an example from Kimura and Ohta (1971): "To carry out mutant s u b s t i t u t i o n at the above rate, each parent must leave 180 78 e =10 o f f s p r i n g f o r only one of the o f f s p r i n g to survive". In humans the average heterozygosity per locus f o r 91 l o c i i s about 8% (Harris and Hopkinson, 1976). The neutral argument i s that the mortality necessary to maintain that much v a r i a t i o n at every locus i s greater than i s ever possible f o r humans; therefore, the observed heterozygosity must be due to some non-selective process. In h i s e a r l i e r attempts to^deal with, the same problem, Fisher (1932) a r r i v e d at a very s i m i l a r conclusion. He recognised a paradox (one might characterise t h i s whole problem as Mendel's paradox) a r i s i n g from the random mating process. On the one hand a large number of new genotypes a r i s e from Mendelian segregation, while on the other hand the phenotype of the randomly mating, population maintains the same d i s t r i b u t i o n . Is t h i s uniform phenotype maintained by the s e l e c t i v e removal of those genotypes that would change the phenotype d i s t r i b u t i o n ? Fisher's (1932) answer i s no and yes. He regarded a l l e l i c variants as being almost p h y s i o l o g i c a l l y i d e n t i c a l . I f the uniform phenotype a r i s e s from a l l the genotypes being v i r t u a l l y p h y s i o l o g i c a l l y i d e n t i c a l , how does s e l e c t i o n work? Fisher (1932, pl6) answers with a curious argument based on an analogy with the 'psycho-p h y s i c a l ' perception of differences i n the weight of objects. This argument concludes: "The s e l e c t i v e advantage w i l l increase or decrease continuously, even f o r changes much smaller than, those appreciable to our own senses, or to those of the predator or other animal, which may enter into the b i o l o g i c a l s i t u a t i o n concerned." At f i r s t sight t h i s statement does not make any l o g i c a l sense. 27 How can s e l e c t i o n operate on such i n v i s i b l e differences? The key to understanding both. Kimura's and Fishery's explanations of a l l e l i c v a r i a t i o n (Fisher (1932). r e f e r s to 'allelomorphs') l i e s i n the kind of biochemical machinery that they were modelling. Unfortunately, neither Kimura (1960) nor Fisher t e l l us i n p l a i n words what sort of biochemistry they had i n mind, but t h e i r biochemistry can be i n f e r r e d from t h e i r models. I t i s c l e a r that they envisioned enzyme functions as operating independently of each other and having l i n e a r and additive net e f f e c t s . A change i n one d i r e c t i o n by one enzyme a l l e l e could be o f f s e t i n a simple additive fashion by another enzyme a l l e l e at another locus. No known biochemistry functions l i k e that. Atkinson (1976) and Hochachka and Somero (1982) c l e a r l y show that biochemical pathways are highly integrated and can be regarded as goal-oriented. The biochemistry of organisms i s not a soup whose flavour can be averaged out over i t s ingredients. Thus the f i g u r e of 8% average heterozygosity per locus i s quite mis-leading. In Figure 2, the number of l o c i i s p l o t t e d against heterozygosity f o r the human a l l e l e data f o r 92 l o c i reported by Harris and Hopkinson (1976). I t i s evident that most l o c i (61) are monomorphic and that most of the heterozygosity i s c a r r i e d by a few l o c i which, with rare exceptions, have only two a l l e l e s . The argument for the existence of endless possible combinations of a l l e l e s , because of some v a r i a t i o n at every locus, i s therefore quite mis-leading. The use of heterozygosity as an o v e r a l l measure of a l l e l i c v a r i a t i o n i s i n part to blame. The categorisation of the data i n t h i s manner by Kimura (1960)- ensures the consequence of h i s argument. I t i s also misleading to assume that the i n t e r a c t i o n s between a l l l o c i 28 F i g . 2 The frequency of l o c i p l o t t e d against heterozygosities (Hz) from 0 to 0.5 f o r 96 enzymes described f o r humans (from Harris and Hopkinson, 1976). 80 O o 40 30 are l i n e a r and a d d i t i v e . I t i s i r o n i c that i n the same issue of the Journal of Heredity as the paper by Kimura (1960) there i s a paper by G i l b e r t (1960) demonstrating experimentally that gene e f f e c t s are not l i n e a r and not a d d i t i v e . Spickett and Thoday (1966) confirmed t h i s n o n - l i n e a r i t y and non-additivity i n a much more extensive experiment. The o r e t i c a l genetic models represent only the gene frequency aspect of the larger and more complex i n t e r a c t i o n s of gene frequency, gene product biochemistry and the physiology and behaviour that r e s u l t s from that b i o -chemistry. Motoo Kimura, who was l a r g e l y responsible for the development of the neutral theory, has avoided using the neutral theory as anything other than a t h e o r e t i c a l d e s c r i p t i o n of gene frequency i t s e l f . I t i s possible that when the knowledge of gene product biochemistry and i t s e f f e c t s on physiology and behaviour are better understood i n quantitative terms, then the mathematical base of the neutral theory can be extended out of t h e o r e t i c a l genetics into more complex aspects of population biology. Unfortunately, some f i s h e r i e s g e n e t i c i s t s (e.g., Allendorf and Utter, 1980) have taken the neutral model too l i t e r a l l y . When the kind of s i m p l i f i e d biochemistry required by Kimura's and Fisher's approach has been assumed i n f i s h e r i e s management studies, the use of heterozygosity has allowed f i s h e r i e s g e n e t i c i s t s to e f f e c t i v e l y blank out differences between year classes (or repeated sampling) and e s t a b l i s h a r t i f i c i a l regional a l l e l e frequency averages that discount the often enormous variance within the regions that they are intended to characterise. For example, Grant et a l . (1980), i n a study of the stock i d e n t i f i c a -t i o n of sockeye salmon, report a large amount of year-to-year v a r i a t i o n i n four l o c i , glutamate pyruvate transaminase, phosphoglucomutase, l a c t a t e dehydrogenase, and phosphoglucose isomerase, examined i n f i s h e s from stations within the K a s i l o f River drainage. These four l o c i show an average diffe r e n c e of 7% i n the p r i n c i p l e a l l e l e frequency between samples taken i n 1975 and 1976. The range i n average difference i s from no diff e r e n c e (in four out of twenty samples) to almost 44%. Grant et a l . (1980) dismiss t h i s v a r i a t i o n : "We tested t h i s assumption i n the K a s i l o f River by sampling the same locations f o r two consecutive years and found no s i g n i f i c a n t difference between years". They were able to make t h i s statement by using the heterozygosity c a l c u l a t e d over these four l o c i and 22 others that were v i r t u a l l y monomorphic.. They assert that 'the estimates are based on genetic data which are not subject to d i r e c t environmental influences and which appear to be stable over time". C l e a r l y we require biochemically r e a l i s t i c models of what a l l e l i c v a r i a n t s a c t u a l l y do before we can resolve Mendel's paradox. Even the simplest kind of study, l i k e the interdependence of a l l e l i c v a r i a t i o n at biochemically juxtaposed l o c i l i k e phosphoglucomutase and glucosephosphate isomerase, reveals s u r p r i s i n g l y complicated i n t e r a c t i o n s (Gauldie and Smith, 1982). STATISTICAL LIMITATIONS TO THE USE OF ALLELE VARIATIONS The use of a l l e l e s based on t h e i r p h y s i o l o g i c a l differences rather than on t h e i r frequency d i s t r i b u t i o n s overcomes a number of s t a t i s t i c a l problems. F i s h e r i e s management usage of a l l e l e phenotypes based on p h y s i o l o g i c a l differences involves at l e a s t two parameters. For example, an observation could be that there i s a c o r r e l a t i o n between the a l l e l e genotype and growth, or disease resistance, so that an exact measure of a l l e l e frequency i n a 32 sample i s not so c r i t i c a l . F i s h e r i e s management usage of a l l e l e phenotypes as neutral markers of populations requires not only an exact measure of the a l l e l e frequency i t s e l f , but also an estimate of i t s r e l i a b i l i t y . The problem of s t a t i s t i c a l r e l i a b i l i t y of a l l e l e frequency has been discussed by Lewontin (1974) and F a i r b a i r n (1979) as a l a r g e l y t h e o r e t i c a l problem. However, just as the issue of p h y s i o l o g i c a l differences versus n e u t r a l i t y of a l l e l e differences can be approached experimentally, so can the problem of r e l i a b i l i t y . At l e a s t four sources of error e x i s t , year-to-year v a r i a t i o n , r e l i a b i l i t y of estimation of electrophoretic patterns from gels, the r e l i a b i l i t y attached to a proportion estimated from a small sample drawn from a large population, and f i n a l l y the problem of hidden genetic v a r i a t i o n . The problem of year-to-year v a r i a t i o n was addressed above and w i l l be considered further i n the following paper i n t h i s s e r i e s on t r a n s f e r r i n a l l e l e s . Year-to-year v a r i a t i o n i s common, and the variance that i t introduces can be considerable. The r e l i a b i l i t y of estimation of electrophoretic patterns from gels hinges around two points; the c r i t e r i o n whereby a p a i r of electrophoretic bands are considered to be a thick single band, or two t h i n s i n g l e bands, and the proportion of unresolvable i n d i v i d u a l s within a sample. Gauldie and Johnston (1980) -used the 'naive person' t e s t and found that a number of electrophoretic phenotypes could not be resolved, even a f t e r s o l u b i l i s i n g more.protein. The 'naive person' t e s t i s simply to present the gel to someone who i s not f a m i l i a r with the vagaries of gel patterns and ask them to i n d i c a t e which samples have bands that can be c l e a r l y counted. This type of r i g o r i s p a r t i c u l a r l y important for marine f i s h f o r which breeding experiments cannot be performed. 33 Gauldie and Johnston (1980) report that the proportion of 'unresolve. able' phosphoglucose isomerase patterns of i n d i v i d u a l s from nine stations f o r the species Thyrsites atun had a Poisson d i s t r i b u t i o n with a mean of 0.036, a standard deviation of 0.'19, and standard error of 0.02. I f the undetected, error i n i d e n t i f y i n g a l l e l e s i s at l e a s t as large as the detected e r r o r , then the mean should be twice as large, the variance twice as large, and the standard.deviation, and standard error 0.14 times as large. The consequent range of expected v a r i a t i o n that may be due to chance i s 0.10, which means that only two samples reported by Gauldie and Johnston (1980) fo r Thyrsites atun are s t a t i s t i c a l l y s i g n i f i c a n t l y d i f f e r e n t , the highest and lowest frequencies. I f the confidence l i m i t s are based on the standard deviation then none of the frequencies observed are s i g n i f i c a n t l y d i f f e r e n t from the mean value. The number of unresolvable r e j e c t s i n the more extended sampling reported for Cheilodactylus macropterus i n Gauldie and Johnston.(1980) (Table 2) i s lower, but the maximum range of the frequency of the three a l l e l e s reported i s small, 0.09 to 0.23, so that the s t a t i s t i c a l a n a lysis i s s e r i o u s l y compromised by t h i s source of e r r o r . In addition, heterozygotes are more l i k e l y to be unreadable so that there i s also a p o t e n t i a l bias towards l o s i n g the l e s s common a l l e l e s . The r e l i a b i l i t y of a proportion drawn from a sample when the e n t i r e population s i z e , or even i t s l i k e l y lower l i m i t s , i s known can be estimated N-n from the formula cr 2 = pq. The minimum estimate of population size N.n (Coleman,, pers. comm.) of the spawning aggregate from which the Pegasus Bay sample (JO409) of Cheilodactylus macropterus (Gauldie and Johnston, 1980) was drawn., i s 500,000 f i s h . For a sample siz e of 500 drawn from t h i s population.we have f o r the S a l l e l e a frequency estimate of 0.14, and a standard deviation of 0.015. The minimum differ e n c e between any frequ-34 ency estimate f o r samples of t h i s s i z e (which i s very large) i s thus 0.06. For smaller samples l i k e the Cloudy Bay sample (J0401) of 94, the standard deviation of the S a l l e l e frequency for a population of 500,000 spawners i s 0.039 with a consequent minimum difference of 0.15 i n a l l e l e frequency. These confidence l i m i t s can be c a l c u l a t e d only on the assumption that the sample i s drawn randomly. For a large spawning aggregate of marine f i s h e s , neither spawning nor' sampling can be random because the area over which a p a r t i c u l a r spawning episode takes p l a c e . i s too large. I t may be that t h i s kind of heterogeneity i s e f f e c t i v e l y randomised over a number of years, but l i t t l e evidence has been c o l l e c t e d to e s t a b l i s h random mating in-" f i s h e s . I t i s evident that t r e a t i n g a l l e l e frequency d i s t r i b u t i o n s as a purely s t a t i s t i c a l problem probably requires a much more rigorous approach than i s to be found i n the l i t e r a t u r e dealing with stock separation. Indeed, the p r i o r information necessary to t r e a t the problem of a l l e l e frequencies s t a t i s t i c a l l y i s usually the very information that the s t a t i s t i c a l analyses are intended to provide. Exploiting.the knowledge of p h y s i o l o g i c a l differences between a l l e l e s removes some of these s t a t i s t i c a l problems. More information i s provided by examining r e l a t i o n s h i p s l i k e that between a l l e l i c phenotype and growth rate. S t a t i s t i c a l problems s t i l l remain, but they now centre around the issue of how much, of the v a r i a t i o n i n one f a c t o r can be explained by another. A l l the s t a t i s t i c s employed in.the analysis of a l l e l e frequencies assume that a random sample has been taken. This i s p a r t i c u l a r l y true of multivariate s t a t i s t i c s l i k e genetic distance (Rogers, 1972) and studies l i k e those of Gormann and Renzi (1980) i n which s t a t i s t i c s based on very small samples ( S 10 ) are used to characterise populations. A random sampling programme i s necessary to e s t a b l i s h such s t a t i s t i c a l bounds of a population, but the only way that a random sampling programme can be devised i s when the population, bounds are already known. For example does a g e n e t i c a l l y i s o l a t e d population comprise 50, 500, 5000, 50,000, or 500,000 f i s h ? "Hidden" a l l e l i c v a r i a t i o n has been reported at a number of l o c i . Hidden a l l e l e s are detected by removing, by denaturation, electromorphs that migrate at the same speed, or by a l t e r i n g the b u f f e r chemistry or pH. "Denaturation"-type, "hidden" a l l e l e s are described by Thatcher . (1977) , Johnson (1978) , Coyne and Felton (1977) and Kreitman (1980). From a n e u t r a l i s t point of view any i n h e r i t e d v a r i a t i o n that has no physio-l o g i c a l consequence provides information about g e n e t i c a l l y i s o l a t e d stocks. But i f these "hidden" a l l e l e s represent p o s t - t r a n s l a t i o n a l modification, then some kind of functional differences might be suspected. 36 CONCLUSION The aim of t h i s paper i s to show that i r r e s p e c t i v e of the conclusions of t h e o r e t i c a l arguments i n genetics, experimental evidence e x i s t s f o r p h y s i o l o g i c a l differences between a l l e l e s . The information that these p h y s i o l o g i c a l differences provide discounts the assumption of n e u t r a l i t y that i s widely accepted i n f i s h e r i e s - o r i e n t e d biochemical studies. This i s not to say that the s t a t i s t i c a l analysis of a l l e l e frequency d i s t r i b u t i o n s i s of no value to.managers. But, i f the b i o l o g i c a l properties of a l l e l e s are ignored, much valuable information r e l a t i n g to the conservation and wise u t i l i s a t i o n of f i s h e r i e s i s l o s t . A f u l l e r appreciation of the f i s h e r i e s management consequences of a non-neutral approach can be drawn from the next two papers i n t h i s s e r i e s . The f i r s t of these deals with variants at the t r a n s f e r r i n locus and the second with the r e l a t i o n s h i p between phosphoglucomutase a l l e l i c phenotypes and growth i n the t a r a k i h i (Cheilodactylus macropterus). 3!7 ACKNOWLEDGEMENTS I am g r a t e f u l to Professors Judy Myers, Don McPhail and Peter Larkin of the U n i v e r s i t y of B r i t i s h Columbia, Dr T. Mulligan and Dr H. 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"Ayu", Plecoglossus  a l t i v e l i s , a f i s h c l o s e l y r e l a t e d to Salmonidae. B u l l e t i n of the Japanese  Society of S c i e n t i f i c F i s h e r i e s 45: 401-406. 5;8 THE ROLE OF ALLELIC VARIATION IN THE MANAGEMENT OF FISHES I I . Limits to the Use of Variants at the T r a n s f e r r i n locus R.W. GAULDIE* *F i s h e r i e s Research D i v i s i o n P 0 Box 297 Wellington, New Zealand New Zealand Research Advisory Council Fellow at The I n s t i t u t e of Animal Resource Ecology The University of B r i t i s h Columbia ;59 ABSTRACT The p h y s i o l o g i c a l r o l e of t r a n s f e r r i n and the biochemical basis of i t s physiology are b r i e f l y described. Evidence f o r the c o n t r o l of a l l e l i c v a r i a t i o n of t r a n s f e r r i n through p h y s i o l o g i c a l differences between a l l e l e s i n vertebrates, including f i s h e s , i s reviewed. The consequences of the adaptive nature of t r a n s f e r r i n v a r i a t i o n are discussed with p a r t i c u l a r reference to year-to-year v a r i a t i o n i n a l l e l e frequency. I t i s argued that the e f f e c t of t h i s year-to-year v a r i a t i o n severely reduces the usefulness of the t r a n s f e r r i n locus i n population or stock studies, and, by extension, the use of a l l e l i c variants of any p r o t e i n f o r measures of stock or population discrimination requires very large samples i n time and space. 60 INTRODUCTION Tr a n s f e r r i n polymorphisms were amongst the f i r s t employed i n f i s h e r i e s management studies to define, "stocks" of f i s h (Moller and Naevdal, 1966). Subsequently, t r a n s f e r r i n s have been used widely f o r genetic distance model-l i n g (Jamieson and Turner, 1976) and for estimating proportions of stocks i n the catch (Fa i r b a i r n , 1979). The use of t r a n s f e r r i n v a r i a t i o n i n f i s h e r i e s management studies has followed the almost universal trend to use a l l e l e frequencies as a s t a t i s t i c a l t o o l to resolve problems associated with the pseudo-taxonomic issue of whether there are g e n e t i c a l l y i s o l a t e d sub-populations within a species. Genetic i s o l a t i o n i s Cushing's (1964) c r i t e r i o n of a stock. In p u r s u i t of these pseudo-taxonomic issues, f i s h e r i e s population g e n e t i c i s t s have generally assumed that there are no p h y s i o l o g i c a l l y s i g n i -f i c a n t d ifferences between a l l e l e s . However, there e x i s t s a substantial l i t e r a t u r e describing p h y s i o l o g i c a l l y important differences between trans-f e r r i n a l l e l e s i n many vertebrates. In t h i s paper .1 review t h i s l i t e r a t u r e and show that the n e u t r a l i s t assumptions about t r a n s f e r r i n v a r i a t i o n cannot be sustained. The s i g n i -ficance of p h y s i o l o g i c a l l y d i f f e r e n t t r a n s f e r r i n a l l e l e s to the current s t a t i s t i c a l usage of t r a n s f e r r i n a l l e l e frequencies i s discussed. 61 METAL ION TRANSPORT AND THE ROLE OE TRANSFERRIN IN DISEASE RESISTANCE T r a n s f e r r i n has two metal binding s i t e s that form complexes with i r o n (Zapolski, Ganz and P r i n c i o t t o , 1974), zinc ( P h i l l i p s , 1976; Evans, 1976), copper (Aasa and Aisen, 1968), and chromium, manganese and cobalt (Aisen, Aasa and Redfield, 1969). There are, i n addition to serum t r a n s f e r r i n s , other t r a n s f e r r i n s associated with the e p i t h e l i a l c e l l s of the gut mucosal membrane (Finch, 1975), i n milk, and i n avian eggs (Feeney and A l l i s o n , 1969) . Although, t r a n s f e r r i n s carry many metals, i t i s i r o n transport by serum t r a n s f e r r i n s that has been most i n t e n s i v e l y studied i n humans and farm animals. Iron binding requires a bicarbonate ion (Van Snick, Masson and Heremans, 1973) and a strong p r o t e i n - i r o n bond i s formed inv o l v i n g 4 nitrogen and 2 or 3 oxygen ligands (Aasa and Aisen, 1968). Thus> the attachment of i r o n to the molecule involves very strong intermolecular forces. Each t r a n s f e r r i n molecule has two i r o n binding s i t e s , each carrying one i r o n molecule. There are thought to be fu n c t i o n a l differences between the binding s i t e s such that one s i t e c a r r i e s i r o n s p e c i f i c a l l y to the developing erythron i n the bone marrow while the other s i t e releases i r o n i n t r a n s i t to polymorphonucleocytes i n the blood (Morgan and Baker, 1969; Hahn, 1973; Ganz and P r i n c i o t t o , 1974;. Chipman and Brown, 1975; Lane •1975; P r i n c i o t t o and Zapolski, 1975; P h i l l i p s , 1976)..' The release of i r o n to the polymorphonucleocyte i s c r i t i c a l to the b a c t e r i o c i d a l r o l e of these c e l l s , perhaps through the i r o n a c t i v a t i o n of myeloperoxidases (Chandra, 1973). The release of i r o n involves the bind-ing of the t r a n s f e r r i n to the polymorphonucleocyte and i s thus dependent on the conformation of the t r a n s f e r r i n molecule (Lane., 1972) . Thus, the 62; transport of i r o n to the b i o l o g i c a l l y appropriate s i t e , and the success of competition for t h i s i r o n by pathogens, i s highly dependent on the shape and surface charge of the t r a n s f e r r i n molecule. Indeed, i t has been shown that although t r a n s f e r r i n s are large molecules r e l a t i v e to i r o n (76,000:56), i r o n saturation of the t r a n s f e r r i n by two i r o n molecules causes the t r a n s f e r r i n to both expand s l i g h t l y and become more spherical by a factor of 25% (Rossemeau-Motreff et a l . , 1971) . Iron withdrawal by the host as a defence against invasions by p a r a s i t e s , p a r t i c u l a r l y protozoans and b a c t e r i a , i s well established (Weinberg. 1971, 1975; Ganzoni and Pushmann, 1975). T r a n s f e r r i n with, i t s highly s e l e c t i v e i r o n r e l e a s i n g properties assumes a . p a r t i c u l a r l y important r o l e i n t h i s defence mechanism by denying i r o n to invading pathogens (Bullen, Rogers and G r i f f i t h s , 1978; Calver, Kenny and Kushner, 1978; Leyland et a l . , 1979). PHYSIOLOGICAL DIFFERENCES BETWEEN TRANSFERRIN ALLELES The conformation.and surface charges of t r a n s f e r r i n s are c r i t i c a l i n t h e i r p h y s i o l o g i c a l r o l e of t r a n s f e r r i n g i r o n to the developing erythron and r e t i c u l o c y t e while at the same time denying the i r o n receptors of b a c t e r i a access to free i r o n . Therefore, one might expect electrophoretic variants, which we know to d i f f e r i n charge state, to also d i f f e r i n t h e i r physiologies. A review of the l i t e r a t u r e confirms t h i s expectation. Birds The wouhd-healing properties of egg white have a long h i s t o r y i n f o l k medicine, and t r a n s f e r r i n , as conalbumin, was f i r s t i s o l a t e d as a b a c t e r i o -c i d a l agent from egg white (Schade and Caroline, 1944). A p a r t i c u l a r l y c l e a r case of pathogen resistance dependence on t r a n s f e r r i n a l l e l e s has been described i n the pigeon,.Columba l i v i a , by F r e l i n g e r (1972) who f i r s t 63 showed an i n v i t r o d i f f e r e n t i a l i n h i b i t i o n of microbial growth by d i f f e r e n t t r a n s f e r r i n a l l e l e s i s o l a t e d from pigeons' egg white. The same e f f e c t was described f o r other t r a n s f e r r i n s by Calver et a l . (1978) . F r e l i n g e r '.(1971) also showed that d i f f e r e n t t r a n s f e r r i n genotypes lead to d i f f e r e n t i a l s u r v i -v a l of squabs due to both t h e i r own, and maternally derived, t r a n s f e r r i n s . When the structures of pigeon A and B t r a n s f e r r i n s were compared by peptide mapping, only a single amino a c i d d i f f e r e n c e was found; asparagine residue i n one a l l e l e substituted f o r a serine residue i n the other (Frelinger, 1973)'. Geographic'and species v a r i a t i o n i n t r a n s f e r r i n polymorphisms i n the family Columbidae were reported.by Ferguson (1971). Feeney and A l l i s o n (1969) described t r a n s f e r r i n v a r i a t i o n for a number of b i r d species. Petrovsky e t a a l . (1973) described a t r a n s f e r r i n polymorphism i n the egg white of Cuban chickens. Brown and Sharp (1970) reported an association between t r a n s f e r r i n phenotype and glutathione l e v e l s i n the blood of pigeons s i m i l a r to the r e l a t i o n s h i p between t r a n s f e r r i n genotype and glutathione l e v e l s described f o r monkeys :(Brown et a l . , 1970). Mammals ^Tran s f e r r i n i s highly polymorphic i n sheep (Fesus and Rasmusen, 1971; S t r a t i l , 1973) and c a t t l e (Komatsu, 1979), and l e s s so i n goats (Watanabe and Suzuki, 1973) and pigs. In pigs, there i s evidence for the t r a n s f e r r i n locus being associated with an early l e t h a l factor i n farrowing (Christensen, 1970; Imlah, 1970). In sheep there i s evidence of a complete i n t e r a c t i o n between t r a n s f e r r i n v a r i a t i o n and reproduction (Khattab et a l . , 1964; Rasmusen:- and Tucker, 1973; Rasmusen:/ 1976) . T r a n s f e r r i n genotypes have been shown to be r e l a t e d to f e r t i l i t y and p r o d u c t i v i t y i n c a t t l e (Kushner et a l . , 1973; Fowle et a l . , 1967). Neethling and Osterhoff (1967) showed that there i s a large d i f f e r e n t i a l i n 64 the i r o n carrying capacity (that i s , a f f i n i t y f o r i r o n under standard condi-tions) iof.fthe'three-imost common ; c a t t l e o a l l e l e s " in-the ratio; (Tf EE, ;l . ,Q:TfAA, 2 TfDD,.1.7). C a t t l e t r a n s f e r r i n also shows abnormalities i n the electropho-r e t i c m o b i l i t y of a l l e l e s due to changes i n the number of surface s i a l i c a c i d residues (Spooner and Baxter, 1969; S t r a t i l and Spooner, 1971). Further examination of t h i s abnormality revealed a s i a l i c a c i d attaching enzyme that i s i t s e l f polymorphic (Spooner et a l . , 1977). E l e c t r o p h o r e t i c , v a r i a t i o n i n mouse t r a n s f e r r i n s following i n f e c t i o n by R i c k e t t s i a mooseri may be due to the attachment of s i a l i c a c i d residues to t r a n s f e r r i n (Awaadet a l , 1978). S i a l y l a t i o n i s also involved.in electro-., phoretic v a r i a t i o n of t r a n s f e r r i n s i n sheep ( S t r a t i l , 1973). T r a n s f e r r i n polymorphisms have been described i n horses (McGuire, 1980), monkeys (Goodman and Wolf, 1963; Goodman et a l . , 1965; Brown et a l . , 1970), A u s t r a l i a n hopping mice (Baverstock, 1976), and f o r seals (Bogdanov, 1977), but without reference to f i t n e s s d i f f e r e n c e s . There i s evidence of d i f f e r e n t i a l mortality between t r a n s f e r r i n a l l e l e s i n mice and voles (Ashton and Dennis, 1971; Gaines et a l . , 1971; Krebs et a l . , 1973). B i r d s a l l (1977) demonstrated an apparent excess of heterozy-gotes at the t r a n s f e r r i n locus i n voles. In humans, a number of t r a n s f e r r i n a l l e l e s have been described with apparently very few amino acid differences between them (Wang and Sutton, 1965; Wang et a l . , 1966; Thymann, 1978; Hoste, 1979; Kuhnl et a l . , 1979; S t i b l e r , 1979). Models f o r t r a n s f e r r i n v a r i a t i o n have been complicated by the discovery of."concealed" a l l e l e s by i s o - e l e c t r i c focussing. In t h i s way the formerly most common a l l e l e s TfC, TfB and TfD are now subdivided i n t o f a m i l i e s of a l l e l e s (Kuhnl et a l . , 1979). Since the discovery that the TfC a l l e l e , the most common a l l e l e i n .65 European populations (0.98), i s a c t u a l l y 3 a l l e l e s with frequencies of 0.79, 0.15 and 0.04, i t has become possible to t e s t for c o r r e l a t i o n s with various diseases. Beckman et a l . (1980) have demonstrated a s i g n i f i c a n t c o r r e l a t i o n between the incidence of the T f C 2 a l l e l e and spontaneous abortion. Fishes The analysis of the r o l e of t r a n s f e r r i n s i n the resistance of f i s h to b a c t e r i a l diseases has been undertaken only recently. The r e s u l t s so f a r are consistent with what i s known i n other organisms: that there i s a dif f e r e n c e i n disease resistance between a l l e l i c v ariants r e l a t e d to the p h y s i c a l properties of the t r a n s f e r r i n molecule (Hershberger et a l . , 1975; Pratschner, 1977; Suzomoto et a l . , 1977; Hershberger, 1978). I t i s not c l e a r how these t r a n s f e r r i n properties i n t e r a c t with the other substances involved i n the natural resistance of f i s h to disease (see Ingram, 1980). Winter et a l . (1980) reported an attempt to extend the e a r l i e r study of t r a n s f e r r i n r e l a t e d disease resistance by Suzomoto et a l . (1977) to include a general genetic e f f e c t . They did t h i s by challenging f i s h from d i f f e r e n t stocks with b a c t e r i a l kidney disease and v i b r i o s i s pathogens. Unlike the e a r l i e r study, t h e i r r e s u l t s show l i t t l e t r a n s f e r r i n r e l a t e d e f f e c t . However, t h e i r experimental design i s not c l e a r l y described, but seems to imply that the coho salmon i n the study were challenged at 12.2°C, rather than 9.7°C and that the steelhead trout were challenged at 17.7°C. I t i s d i f f i c u l t to i n t e r p r e t the d i f f e r e n c e between these r e s u l t s since the f i r s t study (Suzomoto et a l . 1977) also involved i r o n augmentation i n the f i s h studied. The high temperatures employed i n the second experiment may mean no more than that the t r a n s f e r r i n component of disease resistance i s impaired by heat s t r e s s , as i s any other p h y s i o l o g i c a l function (Fryer et a l . , 1976). More generalised r e l a t i o n s between t r a n s f e r r i n s and general condition 66. occur i n f i s h i n a fashion s i m i l a r to that described above f o r domestic and farming stock (Balakhnin and Galagan, 1972; McIntyre and Johnston, 1977; R e i n i t z , 1977). In spite of t h i s evidence for p h y s i o l o g i c a l differences between at l e a s t the most common a l l e l e s , most population genetics studies on f i s h e s have assumed that the v a r i a t i o n i s neutral. Consequently the t r a n s f e r r i n v a r i a t i o n i s usually e i t h e r simply described without comment, or i s assumed to have a simple stochastic d i s t r i b u t i o n and used as a s t a t i s t i c a l t o o l i n separating 'stocks', or even sub-species of f i s h (Moller and Naevdal, 1966;. Barrett and Tsuyuki, 1967; Payne, C h i l d and Forrest<; 1971; Chen and Tsuyuki, 1972; Wright and Atherton, 1972; Wilkins, 1972a, b; Elson, 1973; Morgan, Koo and Krantz, 1973; Utter, Ames and Hodgins, 1973; Payne, 1974; Tsuyuki, Roberts and Best, 1974; G a l l et a l . , 1975; McKenzie and Martin, 1975; C h i l d , Burnell and Wilkins, 1976; Jamieson and Turner, 1976; Ney and Smith, 1976; Krueger and Menzel, 1979). A small number of studies have examined the biochemistry of f i s h t r a n s f e r r i n s (de Ligny, -1968; Aisen, LiebmannV'and.Sia;, .1-97-2;., Her.shb6isg.er:, 1975; Valenta et a l . , 1976, 1977). Valenta et a l . (1976) found that the e l e c t r o -phoretic m o b i l i t i e s of some a l l e l e s depend on the presence of s i a l i c a c i d residues on the surface of the t r a n s f e r r i n molecule that p a r a l l e l s the e f f e c t i n c a t t l e (Spooner and Baxter, 1969: S t r a t i l and Spooner, 1971) and may account f o r the age-dependent changes i n t r a n s f e r r i n patterns described by de Ligny (1968). DISCUSSION Tr a n s f e r r i n a l l e l e frequency data have been applied to f i s h e r i e s management problems i n two ways: the i d e n t i f i c a t i o n of stocks (and even sub-species, the stock i n extremis), and the proportions of stocks i n cat-ches taken at sea or i n some other areas of mixed stocks. While t h i s has been done with other protein a l l e l e frequencies, t r a n s f e r r i n v a r iants, because of t h e i r comparatively well established biochemistry and physiology, provide a good t e s t of the v a l i d i t y of these a p p l i c a t i o n s . The use of any a l l e l i c v ariants i n separating stocks requires at l e a s t one assumption: the a l l e l e frequency d i s t r i b u t i o n must be stationary and have a low variance. I f the d i s t r i b u t i o n of a l l e l e s f l u ctuates r a p i d l y , stock i d e n t i f i c a t i o n i s impossible. This assumption further implies that the a l l e l i c v a r i a t i o n i s i n accordance with the neutral theory and that a l l e l e frequencies w i l l not s h i f t as a r e s u l t of differences i n s u r v i v a l or reproduction between i n d i v i d u a l s due to the a l l e l e s that they carry. One of the consequences of the p h y s i o l o g i c a l differences between a l l e l e s i s that a l l e l e frequencies should vary over time with changes i n the environment and.with disease-related processes or population density. Temporal v a r i a t i o n i n t r a n s f e r r i n a l l e l e frequencies has been reported f o r P a c i f i c h a l i b u t (Tsuyuki, Roberts and Best, 1969) and the A t l a n t i c cod (Jamieson and.Turner, 1976) (Table 1). The range of v a r i a t i o n of frequency i n both studies i s . from very low values to about 0.15. This i s within t h e 2 range of variation, i n a l l e l e frequency reported f o r d i f f e r e n t year classes and length classes of other l o c i (Siebenaller, 1976; Smith, 1979; Gauldie and Johnston, 1980; Grant et a l . , 1980). The v a r i a t i o n s i n frequency of two t r a n s f e r r i n a l l e l e s B2 and C3 reported by Jamieson and Turner (1976) are shown i n Table l : c . For a l l e l e B2, frequencies ranged from 0.12 to 0.48, and f o r the C3 a l l e l e from 0.20 to 0.72. Differences between frequencies of samples taken at d i f f e r e n t times from the same s t a t i o n ranged from 0.05 to 0.12. How much of the Table 1. Time dependent v a r i a t i o n i n t r a n s f e r r i n a l l e l e frequency. a) V a r i a t i o n i n P a c i f i c h a l i b u t , Hippoglossus stenolepis, from Tsuyuki, Roberts and Best, 1969. Tf A TfB TfC A l i t a k Bay 1967 .025 .69 .283 A l i t a k Bay 1968 .030 .70 .268 Kayak Is. 1967 .025 .72 .254 Kayak Is. 1968 .030 .73 .240 She l i k o f f Bay 1967 .038 .64 .327 She l i k o f f Bay July 1968 .020 .65 .330 Shelikoff Bay August 1968 .086 .65 .267 Percentage change i n a l l e l i c frequency measured on the f i r s t year of observation. Tf A TfB TfC A l i t a k Bay 67-68 21 1.4 5.5 Kayak Is. 67-68 18 1.3 5.6 Shel i k o f f Bay 67-68 July 47 2.0 1.0 Shelikoff Bay 67-68 August 127 1.5 18.0 mean percent year-to-year change for TfA 53.0 + 2.51 mean percent year-to-year change for TfB 1.6 + 2.30 mean percent year-to-year change for TfC 7.5 + 2.73 expected maximum observed range expected maximum observed range year-to-year year-to-year range." around TfA around TfA range around TfB around TfB mean .036 i s mean .036 i s mean .680 i s mean .680 i s 0 -> .072 0.020. -+ .086 0.665 -> .690 0.640 -* .730 expected maximum year-to-year range around TfC mean .280 i s 0.220 -> .340 observed range around TfC mean .280 i s 0.240 .330 b) V a r i a t i o n i n the A t l a n t i c cod, Gadus morhua, from Jamie sampled at two or more d i f f e r e n t times. Tf A l A2 B l B2 B3 2N F y l l a s Bank 1966 388 .003 . .110 .173 F y l l a s Bank 1972 224 .103 .219 .004 F y l l a s Bank 1973 140 .086 .221 Danes Bank 1972 60 .100 .120 Danes Bank 1973 196 .130 .240 Nanortahk 1972 142 .130 .120 Nanortahk 1973 404 .100 .150 Farewell 1966 402 .140 .140 .007 Farewell 1972 152 .160 .190 R i t t u Bank 1971 210 .005 .360 .009 R i t t u Bank 1975 208 .004 .480 .004 Grand Bank 1967 320 .003 .460 Grand Bank 1971 198 .370 .010 and Turner (1976) f o r stations that were B4 CI C2 C3 C4 DI D2 006 002 004 .018 .004 .007 .030 .007 .010 .020 .013 .210 .144 .190 .100 .015 .002 .002 .019 .019 .013 .040 .616 .592 .610 .720 .570 .640 .630 .620 .570 .270 .202 .230 .320 .003 .064 .076 .070 .070 .040 .098 .090 .060 .070 .110 .110 .097 .120 .004 .007 .004 .020 .040 KO c) Percentage v a r i a t i o n of the p r i n c i p a l a l l e l e s B2 and C3 i n the A t l a n t i c cod, Gadus morhua, based on the f i r s t year sample (from Jamieson and Turner, 1976). *1 O a 'hd » Q PJ (1) PJ H- K M 3 3 H rt p) H PJ O CD rt 3 p) CO l-i S| C Qi co rt n> W Oi M W W rjfl pj tr h-1 pi oi ft) 3 3 3 3 * W M M i-> i- 1 H M r-1 H M M M r-> VO VO VD VD VD VD VD VD VD VD VO VD VD ~J ~J ~J -~J CTi ~J - J »J CTi - J CTl CO OJ CO OJ CO OJ CTl CO M Ul -o M B2 .17 .22 .22 .12 .24 .12 .15 .14 .19 .36 .48 .46 .37 B3 .62 .59 .61 .72 .57 .64 .63 .62 .57 .27 .20 .23 .32 Frequency Change on base year % B2 29 29 100 25 36 33 20 Change on base year % C3 4.8 1.6 21 1.5 8 26 39 7'1 t o t a l v a r i a t i o n can be explained by the time-dependent v a r i a t i o n of the B2 and C3 a l l e l e frequency? The number of duplicate samples i s too low to allow an analysis of variance, but an estimate of the contribution of the year-to-year v a r i a t i o n to the o v e r a l l v a r i a t i o n can be made i n the following way. The o v e r a l l v a r i a t i o n of the B2. a l l e l e frequency has a mean value of 0.25 and a standard deviation (assuming a normal d i s t r i b u t i o n ) of 0.13 for a l l 13 observations. The change.in B2 a l l e l e frequency from year to year measured on. the frequency of the f i r s t year of sampling ranged from 20 to 100% (Table l . c ) , with a mean of 39% and a standard deviation of 27% for the seven observations. The expected range of percentage changes from year to year i s then 39± 2x27%, i . e . , from 0 to 93% of the o v e r a l l mean. Since the percentage change i s measured absolutely, the maximum expected range of the o v e r a l l mean i s from 0.25 -(.93x0.25) to 0.25 +(93x0.25), that i s 0.018 to 0.483, while the minimum expected range i s no change at a l l . I t can be seen that the maximum expected range accommodates the highest and lowest B2 frequencies, 0.48 and 0.12 re s p e c t i v e l y . The o v e r a l l v a r i a t i o n of the C3 a l l e l e frequency has a mean value of 0.51 and a standard, deviation (assuming a normal d i s t r i b u t i o n ) of 0.18 for a l l 13 observations. The change i n C3 a l l e l e frequency from year to year measured on the frequency of the f i r s t year of sampling ranges from 1.6 to 39% (Table l.c) with a mean of 15% and a standard deviation of 14% for the seven observations. The expected range of percentage changes from year to year i s then 15± 2x14%, i . e . from 0 to 43%. Consequently the expected year to year v a r i a t i o n i s from 0 to 43% of the o v e r a l l mean. Since the percent-age change i s measured absolutely, the maximum expected range of the o v e r a l l mean i s from 0.51-(.43x.51) to 0.51 + (.43x.51), that i s 0.29 to 0.73, while 72 the minimum expected range i s no change at a l l . The highest and lowest observed C3 frequencies, 0.72 (Danas Bank) and 0.20 (Rittu Bank), are s u f f i c i e n t l y f ar apart to suggest that the difference between them i s greater than that expected from the year-to-year v a r i a t i o n . But the other observations made at these'two sta t i o n s , 0.57 and 0.27, are very close to the expected year-to-year range i n v a r i a t i o n . Time-dependent v a r i a t i o n i n the P a c i f i c h a l i b u t i s not as great as that for the A t l a n t i c cod, but, nonetheless, a crude estimate of variance based on the l i m i t e d sample siz e shows enough time-dependent v a r i a t i o n to accom-modate the maximum differences i n a l l e l e frequency for the TfA and TfC v a r i a t i o n from d i f f e r e n t regions .reported, but not for the differe n c e i n TfB v a r i a t i o n . However, the sample sizes f or the two samples with maximum differen c e (Kayak Is. , 1968 and.Shelikoff Bay, 1967) are such that the differences between them are not s t a t i s t i c a l l y s i g n i f i c a n t . Kayak Is., 1968, 0.73, sample siz e 100;. Sh e l i k o f f Bay, 1967, 0.64, sample siz e 66; Z s t a t i s t i c = i:23, while z 1 Q = 1.645. A l l t h i s v a r i a t i o n i s reported from samples composed of aggregates of year classes. A l l the samples reported i n Tsuyuki, Roberts and Best (1969) and Jamieson and Turner (1976) are shown to be i n Hardy-Weinberg e q u i l i -brium. However, other studies have shown that aggregates of d i f f e r e n t year classes may be i n apparent Hardy-Weinberg equilibrium even though year classes have s i g n i f i c a n t differences i n a l l e l e frequency (Gauldie and Johnston, 1980; Smith, 1980). Indeed Smith (pers. comm.) has shown that these i n d i v i d u a l year classes, although they are themselves' i n Hardy-Weinberg equilibrium, can be divided into length classes that have s i g n i -f i c a n t a l l e l e frequency differences, and yet these separate length classes also remain i n Hardy-Weinberg equilibrium. 73 I f the Hardy-Weinberg e q u i l i b r i a reported by Tsuyuki, Roberts and Best (1969) and Jamieson and Turner (1976) are more apparent than r e a l , then i t i s p ossible that averaging the frequencies of d i f f e r e n t year classes conceals s t i l l more v a r i a t i o n . Regional differences i n a l l e l e frequency s t i l l no doubt e x i s t even with t h i s large year-to-year v a r i a t i o n . But detecting t h i s l o c a t i o n e f f e c t above_the year-to-year noise i s not possible without an orthogonal experi-mental design which i s very often extremely d i f f i c u l t to arrange i n sampling a f i s h e r y . Not only does the weather impede such rigorous sampling, but the population density and age structure v a r i e s from l o c a t i o n to l o c a t i o n . In addi t i o n , a sample of f i s h taken i n a ground trawl i n 15 minutes i s not comparable to a sample of the same number obtained by exhaustively sampling an a r e a j f o r several days. Ad d i t i o n a l v a r i a t i o n due to the movement of the true; population bounda-r i e s i s no doubt concealed within year classes i n the sample. Thus the v a r i a t i o n reported by Tsuyuki, Roberts and Best (1969) and Jamieson and Turner (1976) i s the sum of the population movement variance and the year c l a s s variance and the r e a l variance of samples may be even greater than we have shown. Thus, the very small differences i n frequency used by Payne et al.(1971) and C h i l d , et a l . (1976) to e s t a b l i s h C e l t i c and boreal races f o r the European A t l a n t i c salmon (0.0015 to 0.0314) must be treated with great suspicion. Without firm evidence f o r the absence of the year-to-year v a r i a t i o n , i t i s evident that the differ e n c e between these "races", when considered i n the knowledge of time-dependent v a r i a t i o n of t r a n s f e r r i n a l l e l e s i n other f i s h e s , i s too small to accept. The use of a l l e l e frequencies to assign proportions of component stocks to mixed stock catches presents a number of s i m i l a r problems. The argument for assigning stock proportions depends on three components: (1) the a l l e l e frequencies of the component stocks; (2) the a l l e l e frequency of the "mixed stock" catch; and (3) the proportions of f i s h from each component stock i n the mixed stock. I f any two of these components are known, then the t h i r d can be predicted. F i s h e r i e s managers have t r i e d to use the a l l e l e frequen-c i e s of the component stocks and the a l l e l e frequency of the mixed stock to p r e d i c t the proportions of component stock f i s h i n the mixed stock. This has been attempted for Columbia River Chinook salmon (Milner and Teel, 1979) Alaskan sockeye salmon, Grant et a l . (il980) both in v o l v i n g many genetic l o c i and A t l a n t i c salmon, using i s o c i t r a t e . dehydrogenase and amino aspartate transferase variants (Cross et a l . , 1978); and t r a n s f e r r i n variants (Fai r b a i r n , 1979; F a i r b a i r n and Roff, 1979). The technique can be summarised i n t h i s way. Given a stock A with a l l e l e frequency a of a l l e l e x, a stock B with an a l l e l e frequency b of a l l e l e x, and a sample C that i s a mixture of both stocks with an a l l e l e frequency c of a l l e l e x, then the proportion of stock A to stock B i s given by the r a t i o 1:K from the expression: a K = b.c The r e l i a b i l i t y of t h i s estimate i s the variance of K. The variance of K i s complex, but i s given by the following expansion: s 2, = (1/b.c) 2. s 2 + ( - a / c . ( b ) 2 ) 2 . s 2 K a b + (-a/b,(c) ?) 2 . s 2 c + (1/b.c). (-a/c(b). 2). cov(a,b) + (-a/c(b) 2).. (-a/b. ( c ) 2 ) . cov(b,c) + (1/b.c) (-a/b.(c ) 2 ) . cov(a,c) 75 The covariances are l i k e l y to be negative, but the variance of K w i l l s t i l l be quite large when the year-to-year v a r i a t i o n i s taken i n t o account. Of these stock proportion studies, only one (Cross, et a l . , 1978) attempts to t e s t the necessary stationarity;:..and, low .variance-that t h i s technique requires by looking at time-dependent v a r i a t i o n . The difference i n a l l e l e frequency at the amino.aspartate transferase locus between adult salmon and t h e i r supposed.progeny (as parr) from the River Blackwater i s 0.011. Single observations l i k e t h i s have proven to be very u n r e l i a b l e i n other studies (Chilcote et a l . , 1980). Milner and Teel (1979) and Grant et a l . (1980), i n t h e i r approach to the same problem, present an even more unfavourable data set. Their d e f i n i t i o n of "stocks" encompasses samples of widely d i f f e r e n t a l l e l e frequencies; differences within stocks were as high as 0.08. (Milner and Teel, 1980) and year-to-year differences i n frequency as high as 44% (Grant et a l . , 1980). The confidence l i m i t s of the estimate of the proportion of component stocks i n Milner and Teel (1980), (there are more than two) i n the mixed stock;.encompass zero and one. Another aspect of t h i s problem l i e s i n the recognition of a "mixed stock" sample. F a i r b a i r n (1979) and F a i r b a i r n and Roff (1979) r e l y on the Hardy-Weinberg equilibrium as an adequate t e s t . There are good grounds to doubt whether t h i s i s a v a l i d t e s t . I n J t h e J f i r s t place, we have already noted that samples that are l i k e l y to be mixtures, or are known to be mixtures, are often i n Hardy-Weinberg equilibrium. Second, i t has been demonstrated by Lewontin and Cockerham (1959) that the X 2 t e s t i s not very powerful i n comparing observed and expected Hardy-Weinberg proportions of genotypes. Many electrophoretic patterns are not easy to read c l e a r l y , so that there i s always a danger of assigning the wrong i n t e r p r e t a t i o n to the information i n the g e l . Breeding experiments have a l i m i t e d usefulness since c e r t a i n proteins may denature i n s o l u t i o n i n a p a r t i c u l a r sequence, so that there i s always the p o s s i b i l i t y of some bands, and the environment may serve to s h i f t the equilibrium between meric forms of dimers, trimers and tetramers (Yamawaki and Tsukuda, 1979; Tsukuda, 1975). One approach would be to note the d i s t r i b u t i o n of the frequency of samples that could not be resolved into a c l e a r set of bands and use the variance of t h i s d i s t r i b u t i o n as an estimate of the worst possible error. Unfortunately, t h i s kind of t e s t has a c e r t a i n ambivalence due to the differences i n c r i t e r i a of "resolvable" between d i f f e r e n t workers. There are instances of published gel patterns that are v i r t u a l l y unreadable without a great f a m i l -i a r i t y with the t y p i c a l l y observed patterns and great confidence i n the proposed underlying genetic models (Allehdorf .and Utter, 1973; R e i n i t z , 1977; Grant et a l . , 1980). Jamieson and Turner (1976) c a l c u l a t e a number of genetic distance measurements based on the frequencies of the seventeen a l l e l e s of the A t l a n t i c cod t r a n s f e r r i n locus. . They o f f e r no confidence l i m i t s f o r these s t a t i s t i c s , although they use them to structure the populations of A t l a n t i c cod. Confidence l i m i t s of genetic distances are r a r e l y offered i n the l i t e r a t u r e ; even the t h e o r e t i c a l variance of the s t a t i s t i c i t s e l f i s r a r e l y estimated. I t i s evident from the time-dependent v a r i a t i o n we have described that genetic distance measurements are useless unless t h e i r con-fidence l i m i t s are established on the basis of at l e a s t the observed v a r i a t i o n between age classes. . s Genetic distances based on 10 to 20 i n d i v i d u a l s per sample (e.g., Gorman and Renzi, 1979; Buth, Burr and Schenk, 1980) must be regarded with 77 caution. Such studies are v a l i d only with i d e a l random sampling techniques. The design of random sampling experiments requires that the experimenter already knows the bounds of the population that i s being sampled. These bounds are usually the very information that genetic distance s t a t i s t i c s are supposed to provide. So f a r , our discussion has pursued a p e s s i m i s t i c and negative l i n e . This should not be interpreted to mean that a l l e l e frequency data have no place i n f i s h e r i e s management. The problem has been that a l l e l i c v a r i a t i o n has been c a l l e d on to r a t i f y a hypothesis that i s quite u n r e a l i s t i c . There is: nothing i n physiology, ecology, or morphology to suggest that, i n nature, species are divided into p h y s i c a l l y i s o l a t e d , g e n e t i c a l l y homogenous (that i s "pure") s t r a i n s . When we regard f i s h e s as being good (in the sense of Mayr, 1970) species that are broken up into l o c a l and transient genetic types depending on both migration and environment pressure, we discover that a l l e l e data have a much more important, indeed fundamental, r o l e to play i n f i s h e r i e s management that i s not r e l a t e d to the u n r e a l i s t i c , pseudo-taxonomic issue of "stocks". Once i t i s recognised that a l l e l i c v a r i a t i o n i s a part of the Vfine-tuning" of an organism .to i t s environment, then i t can be seen that at l e a s t two management problems, growth and mortality, must be linked to a l l e l i c v a r i a t i o n . Stock-recruit r e l a t i o n s h i p s provide the most formal descriptions of r e l a t i v e m o r t a l i t i e s i n f i s h e r i e s management. Unfortunately, s t o c k - r e c r u i t r e l a t i o n s h i p s s u f f e r from a very high variance that increases with stock density or concentration. Thus, there i s l i t t l e i n s i g h t to be gained by seeking c o r r e l a t i o n s between a l l e l e frequencies at l o c i l i k e t r a n s f e r r i n that we know to be associated with disease - a density dependent mortality 78 factor - and the algebraic parameters of the equation r e s u l t i n g from mean recruitment as a function of. mean stock density. The v a r i a t i o n i n stock-r e c r u i t curves i s too high. However, the v a r i a t i o n at the high stock density part of the. s t o c k - r e c r u i t r e l a t i o n s h i p often has a Poisson d i s t r i b u -t i o n (variances.increase with the mean) that i s suggestive of disease-related processes. By exploring the r e l a t i o n s h i p between the a l l e l i c frequencies of proteins involved i n disease resistance we may be able to show that some of the variance i n the stock r e c r u i t r e l a t i o n s h i p s has a genetic b a s i s . Growth i n fi s h e s provides a much more tr a c t a b l e r e l a t i o n s h i p . The very strong c o r r e l a t i o n between growth curve parameters and a l l e l i c v a r i a -t i o n at the phosphoglucomutase locus i n Cheilodactylus macropterus i s the subject of the next paper i n t h i s s e r i e s . 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THE ROLE OF ALLELIC VARIATION IN THE MANAGEMENT OF FISHES I I I . D i f f e r e n t Growth Curve Parameters Associated with A l l e l i c Phenotypes at the Phosphoglucomutase Locus i n Cheilodactylus macropterus (Cheilodactylidae: Telostei) f R. W. Gauldie *W. J . Gazey t F i s h e r i e s Research D i v i s i o n P 0 Box 297 Wellington, New Zealand New Zealand Research Advisory Council Fellow at The I n s t i t u t e of Animal Resource Ecology The University of B r i t i s h Columbia *L. G. L. Consulting Company Bryan, Texas, U.S.A. ABSTRACT A l l e l i c phenotypes of the l i v e r enzyme phosphoglucomutase are shown to be associated with d i f f e r e n t growth parameter values of the von Be r t a l a n f f y curve f o r the f i s h Cheilodactylus macropterus. Differences i n a l l e l e frequency between exploited and unexploited populations of C_. macropterus may be due to differences i n growth rate of a l l e l e phenotype c a r r i e r s , a m u l t i - a l l e l e "Ricker" e f f e c t generated by f i s h i n g pressure, or to temperature differences during spawning and early development. 98 INTRODUCTION What are the consequences to f i s h e r i e s managers of the observation that differences i n a l l e l e frequency between w i l d populations represent differences i n p h y s i o l o g i c a l adjustment to the environment? Ricker (1973) has pointed out the problems that a r i s e i n f i s h e r i e s management when p h y s i o l o g i c a l differences i n f i s h populations r e s u l t i n differences i n the growth curve parameters that are used i n p r o d u c t i v i t y modelling. Ricker (1973) showed that i n such circumstances f i s h i n g e f f o r t based on the mean growth c h a r a c t e r i s t i c s of a mixed population w i l l drive that population towards a monoculture of the f a s t e s t growing type. I f i n d i v i d u a l s carry-ing d i f f e r e n t a l l e l e s grow at d i f f e r e n t rates as a consequence of p h y s i o l o g i c a l differences between these a l l e l e s , then Ricker's (1973) multi-species problem can be extended to a m u l t i - a l l e l e problem. In a recent study of a l l e l i c v a r i a t i o n i n some New Zealand f i s h e s (Gauldie and Johnston, 1980), length, age and a l l e l i c genotypes at the phosphoglucomutase (PGM) locus were recorded f o r a large number of t a r a k i h i (Cheilodactylus macropterus), a marine f i s h of commercial importance i n the New Zealand trawl f i s h e r y . In t h i s paper we w i l l t e s t the m u l t i - a l l e l e "Ricker" e f f e c t by examining the association between growth curve parameters and PGM a l l e l i c genotypes and the s i g n i f i c a n c e of that association to f i s h e r i e s management. . 99 MATERIALS AND METHODS The d e t a i l s of c o l l e c t i o n and methods of electrophoresis f o r the a l l e l i c data are recorded i n Gauldie and Johnston (1980). The s t a t i o n numbers and notation used by Gauldie and Johnston (1980) are maintained i n t h i s paper. Samples were obtained from three st a t i o n s , J0407, J0409 and J0415, located on the West Coast of the South Island of New Zealand by trawling from the research vessel James Cook. The f i s h were maintained i n a l i v e tank, k i l l e d , l i v e r s removed and frozen i n l i q u i d nitrogen. O t o l i t h s were removed at the same time and the f i s h sexed. The o t o l i t h s were read by transmitted l i g h t following the method of Tong and Vooren (1972). Electrophoresis was c a r r i e d out on c e l l u l o s e acetate plates using Helena HB barbitone b u f f e r at pH 8.1. Non-linear estimation procedure f o r asymptotic length and growth c o e f f i c i e n t parameters f o r von B e r t a l a n f f y curve follows the method of Galucci and Quinn (1979). The length of f i s h at estimated age zero was constrained to zero following the common p r a c t i c e i n t h i s regard. RESULTS 100 G a l l u c c i and Quinn (1979) point out that there i s always a high c o r r e l a t i o n between L r o and k values of. the von Ber t a l a n f f y curve (L^ _ L Q o ( l - e t C >^)) i r r e s p e c t i v e of the estimation procedure (for k and L f o r t o t a l females i n t h i s study the c o r r e l a t i o n i s 0.92) . Consequently i t i s necessary to evaluate the e f f e c t s of differences i n L before d i f f e r e n t k values can be accepted. Galucci and Quinn (1979) do t h i s by f i r s t comparing the, s t a t i s t i c W = L .k. I f differences i n W are s i g n i f i c a n t , then s i g n i f i c a n t differences i n k must be due to the e f f e c t s unrelated to changes i n L .. A comparison by t - t e s t of the W values of t o t a l males and females shows a s i g n i f i c a n t difference between growth c o e f f i c i e n t s , and the consequent t - t e s t of the di f f e r e n c e between growth c o e f f i c i e n t values f o r t o t a l males and females i s also s i g n i f i c a n t (Table l . a ) . Comparisons of W values and growth c o e f f i c i e n t s (k) 1 values f o r the three stations J0407, J0409, and J0415 are given i n Table 1: b,c,d. A l l the W values between males and females at these stations are s i g n i f i c a n t . A complete breakdown i n t o the s i x phosphoglucomutase a l l e l i c phenotype classes MM, MS, MF, FF, FS, and SS i s given f o r male and female growth c o e f f i c i e n t s f o r stations J0407, J0409, and J0415 i n ;Table 1: b,c,d . The s i g n i f i c a n c e of differences i n the W value and the growth c o e f f i c i e n t k by sex and phenotype i s shown (where sample sizes permit) f o r each of the stations J0407, J0409 and J0415 (Table 1: b,c,d). 101 Table 1 Values of the asymptotic length (L^ ), growth c o e f f i c i e n t (k), and the Galucci and Quinn s t a t i s t i c s (W) and t h e i r respective variances are shown f o r males and females by genotype at stations J0407, J0409 and J0415 recorded by Gauldie and Johnston (1980). S t a t i s t i c a l comparisons within and among these parameters are included. The sample s i z e i s given by N and the sums of squares by SS. 102 Table 1- section a (i) T o t a l sample constrained t o ^ t = 0. N = 1656, SS = 21750.8 L = 39.79, .2387 x 10~ 2 0 0 ' k = .3183, .1166 x 10~ 5 W = . .12.6652, .472 x 10~ 4 ( i i ) T otal males i n sample constrained to t = 0. o N = 866, SS = 9574.3 L = 38.67, .374 x 1 0 _ 2 0 0 k = .3282, .234 x 10~ 5 W = 12.6915, .616 x 10~ 2 ( i i i ) T o t a l females i n sample constrained to t = 0. N = 698, SS = 9435.4 L = 42.16, .8307 x 10~ 2 0 0 ' k = .2875, .238 x 10~ 5 W = 12.121, .804 x 10~ 2 For the difference i n W between males and females t = 132. s For the difference i n k between males and females t = 516. s 103 Table 1 section b Values of the Galucci and Quinn s t a t i s t i c W f o r males and females by genotype at s t a t i o n J0407. (i) Tests f o r the s i g n i f i c a n c e of differences between males and females at s t a t i o n J0407. Males Females 2 2 a o Stn. Phen N W w N W w t - t e s t J0407 MM 65 13.219 .185 84 12.666 .151 7.9 J0407 MS 31 13.636 .454 32 12.606 .540 5.8 J0407 MF 6 12.643 1.589 13 13.069 5.097 0.48 ( i i ) Within s t a t i o n tests of the s i g n i f i c a n c e of differences i n the Galucci and Quinn s t a t i s t i c W shows for the s t a t i o n J0407: Males J0407 MM x J0407 MS t = 3.14 s Females J0407 MM x J0407 MS t = 0.44 s ( i i i ) A t e s t f o r the s i g n i f i c a n c e of the difference i n growth c o e f f i c i e n t k between MM and MS males and females at s t a t i o n J0407. 2 2 Stn Pheno N k °k Stn Pheno N k °k t - t e s t -4 -3 Males J0407 MM 65 .321 .644x10 J0407 MS 31 .344 .177x10 8.8 -4 -3 Females J0407 MM 84 .296 .464x10 J0407 MS 32 .297 .168x10 N S 104 Table 1 section c'Values of the Galucci and Quinn s t a t i s t i c W f o r males and females by genotype at st a t i o n J0409. (i) Tests f o r the s i g n i f i c a n c e of differences between males and females at s t a t i o n J0409. Males Females w ( i i ) W 9.834 .055 8.332 .052 Stn. Phen N W w J0409 MM 219 10.780 .026 J0409 MS 71 11.397 .098 J0409 MF 31 9.559 .132 J0409 SS 8 12.803 1.196 Within s t a t i o n tests of the Galucci and Quinn s t a t i s t i c W show: Males Females N 107 40 16 11.932 .292 9 8.367 .347 t- t e s t 41.88 59.77 14.88 10.71 J0409 MM-MS t = 16.04 s J0409 MS-MF t = 23.9 s J0409 MM-MF t =17.9 s J0409 MF-SS t = 8.75 S J0409 MS-SS t = 3.84 s J0409 MM-SS J0409 MM-MS t =33.6 S J0409 MS-MF t =24.2 s J0409 MM-MF t =14.35 s J0409 MF-SS t =14.6 s J0409 MS-SS t = 5.55 s t = .17 s J0409 MM-SS t = 7.14 s ( i i i ) Test of s i g n i f i c a n c e of differences i n the value of the growth c o e f f i c i e n t k between males and females. Stn. Phen N k J0409 MM 219 .279 J0409 MS 71 .299 J0409 MF 31 .299 J0409 SS 8 .363 ~k -3 .107x10 -4 .415x10 -4 .388x10 -4 .702x10 N 107 40 16 9 k .229 .179 .268 .188 ~k -3 .155x10. -3 .119x10 -4 .789x10 -3 .156x10 t - t e s t 115.9 128.5 15.8 17.9 105 Table 1 section d Values of the Galucci and Quinn s t a t i s t i c W f o r males and females by genotype at s t a t i o n J0415. Males Females 2 2 a a Stn Phen N W ow N W w t - t e s t (i) J0415 MM 197 12.27 .135 201 11.76 .154 12.2 J0415 MS 52 13.70 .318 60 11.71 .522 15.3 J0415 MF 27 11.41 1.140 21 10.76 3.09 1.35 ( i i ) Within s t a t i o n tests of the s i g n i f i c a n c e of differences between the values of the Galucci and Quinn s t a t i s t i c W. Males: MM-MS t = 142 Females: MM-MS t =0.48 s s ( i i i ) Tests of s i g n i f i c a n c e of differences i n the value of the growth c o e f f i c i e n t k between males and females. 2 2 Stn Phen N k k N k k t - t e s t J0415 MM 197 .2957 .330xl0~ 4 201 .2578 .296xl0 _ 4 61.8 J0415 MS 52 .368 .109xl0~ 3 60 .257 .104xl0~ 3 58 -3 -3 J0415 MF 27 .254 .228x10 21 .202 .416x10 8.8 (iv) Within s t a t i o n tests of the s i g n i f i c a n c e of differences i n the value of the c o e f f i c i e n t k between d i f f e r e n t phenotypes. Males: MM-MS t =45.5 s Females: MM-MS t_ = .34 Within s t a t i o n tests of the s i g n i f i c a n c e c o e f f i c i e n t k f o r the s t a t i o n J0409: Males J.0409 MM-MS t =24.54 s J0409 MS-MF t =52.5 s J0409 MM-MF t =45.2 s J0409 MF-SS t =15.35 s J0409 MS-SS t = 7.24 s J0409 MM-SS t = 9.45 106' of differences i n the growth Females J0409 MM-MS t = 75 s J0409 MS-MF t =36.4 S J0409 MM-MF t =16.35 s J0409 MF-SS t =16.5 s J0409 MS-SS t = 2.2 s J04Q9 MM-SS t = 9.6 DISCUSSION There i s a se r i e s of questions one can ask of the data. 1. Are there r e a l differences i n growth curve parameters f o r in d i v i d u a l s with d i f f e r e n t a l l e l i c genotypes of PGM? The von Ber t a l a n f f y growth curve c o e f f i c i e n t (k) f o r the ent i r e sample c o l l e c t e d and aged by Gauldie and Johnston (1980) was 0.328 f o r males and 0.288 f o r females. There i s a s i m i l a r sex difference i n growth between males and females at stations J0407, J0409 and JO415, as w e l l as between males and females with the same PGM genotype. S t a t i s t i c a l l y s i g n i f i c a n t differences i n the value of the growth c o e f f i c i e n t (k) occur between males with the MM and MS genotype at s t a t i o n J0407 (Table 1: b), but the differ e n c e between female MM and MS genotype growth c o e f f i c i e n t s at t h i s s t a t i o n i s not s i g n i f i c a n t (Table 1: b). However, there i s a s i g n i f i c a n t difference i n growth rate between male and female MM and MS genotypes (Table 1: b). The number of i n d i v i d u a l s with other genotypes at s t a t i o n J0407 i s too low to t e s t . There are also s t a t i s t i c a l l y s i g n i f i c a n t differences i n the growth c o e f f i c i e n t (k) between males and females with the genotypes MM, MS, MF and SS at s t a t i o n J0409 (Table 1: c ) . S i m i l a r l y , at s t a t i o n JQ415 there are s t a t i s t i c a l l y s i g n i f i c a n t differences i n the growth c o e f f i c i e n t (k) between males and females with the genotypes MM, MS,. MF and SS (Table 1: d) . Thus, the observed differences i n the growth c o e f f i c i e n t (k) are, with few exceptions, greater than expected by chance and are not art e f a c t s of the c o r r e l a t i o n between the growth curve parameter (k) and the asymptotic length L . The data f o r each of the three stations are drawn from a portion of the e n t i r e p o s s i b l e . t a r a k i h i growth curve, so that each population repres-ents a d i f f e r e n t , although overlapping, part of the possible t a r a k i h i growth curve,. Since the populations sampled may never have s i g n i f i c a n t numbers of i n d i v i d u a l s smaller or larger than those i n the samples taken by Gauldie and Johnston (1980), the extent to which t h i s incomplete data generates errors can be assessed only by further experiments. 2. Is there any biochemical evidence that PGM i s associated with growth? PGM i s not a regulatory enzyme i n the usual sense ( i . e . c o n t r o l l e d by feedback). The enzyme i s f r e e l y r e v e r s i b l e with a A G° of about zero and i t has a high t i t r e . The enzyme thus acts as a "hole" through which glucose-6-phosphate (G-6-P) and glucose-l-phosphate (G-l-P) can flow f r e e l y i n e i t h e r d i r e c t i o n depending on the stoichiometry of the G-l-P and G-6-P concentrations. Although PGM i s not a regulatory enzyme i n the usual sense (Stanbury et a l . , 1978), the reaction catalysed by PGM does involve two substrates, G-6-P and F-2-diP, with a strong product i n h i b i t i o n e f f e c t (Ray and R o s c e l l i , 1964), that would permit modulation of the substrate competition with other G-6-P using enzymes. Consequently a l l e l i c variants at the PGM locus with d i f f e r e n t biochemical properties would serve to p a r t i a l l y re-route carbohydrates through e i t h e r glycogen storage, ATP production, or f a t t y a c i d synthesis and thus influence growth rate.-109 3. Do the differences i n growth rate have any s i g n i f i c a n c e to the management of the t a r a k i h i f ishery? Vooren (1973) described the population dynamics and e f f e c t s of f i s h i n g pressure on the t a r a k i h i f o r both the East Cape and the Kaikoura regions. The Pegasus Bay sample (J0409) (Gauldie and Johnston, 1980) was drawn from the Kaikoura region. Vooren (1973) notes that before 1966 the Pegasus Bay (Kaikoura) f i s h e r y was v i r t u a l l y unexploited. Since 1966 the t a r a k i h i has been heavily f i s h e d i n the Pegasus Bay area and f i s h i n g pressure has been maintained i n the East Cape area. Since Gauldie and Johnston (1980) reported a l l e l e genotype frequencies from the 1966 to the 1973 year classes of the Pegasus Bay f i s h e r y , i t i s possible to compare a l l e l e phenotype frequencies from an unexploited year with an exploited year (1966 and 19 73) i n the Pegasus Bay f i s h e r y , and at the same time to compare an exp l o i t e d f i s h e r y (East Cape) with an unexploited population (the B l u f f sample (J0607)). The comparisons are made i n the following table: FF FM MM MS SS FS Unexploited B l u f f , 1977 .02 .04 .62 .27 .04 .0 Unexploited Pegasus Bay, 1963 .03 .07 .62 .24 .03 .0 Ex p l o i t e d Pegasus Bay, 1973 .0 .20 .60 .18 .03 .0 Expl o i t e d East Cape, 1977 .02 .14 .64 .17 .01 .01 The Pegasus Bay samples show an increase i n the proportion of FM a l l e l e phenotype c a r r i e r s and a decrease i n the proportion of MS a l l e l e type c a r r i e r s that p a r a l l e l s the differ e n c e i n a l l e l e phenotype c a r r i e r frequencies between the exploited East Cape and unexploited B l u f f populations. H Q F i g . 1: Sex r a t i o f o r each length group f o r the t o t a l catch of Cheilodactylus macropterus made between A p r i l and J u l y 1967, March and July 1968, and March and June 1969 (from Tong and Vooren, 1972). Length (cm) The growth rates and asymptotic lengths (in cm) of the d i f f e r e n t a l l e l i c genotypes of the Pegasus Bay sample are as follows. Males Females FM 0.226, 42 0.268, 44.5 MS 0.299, 38 0.179, 46 Tong and Vooren (19 72, p. 43) report a difference i n sex r a t i o with length f o r Cheilodactylus macropterus taken i n the trawl f i s h e r y . Their data show that an excess of the small f i s h which are caught are males, and that the l a r g e s t f i s h are v i r t u a l l y a l l females (Fig. 1). Male MS i n d i v i d u a l s are the f a s t e s t growing, so that the catch data i n d i c a t e that MS males are l i k e l y to be depleted i n number as a r e s u l t of f i s h i n g pressure. In addition, the " f i s h i n g down" e f f e c t - the rapid removal of the l a r g e s t a v a i l a b l e f i s h - w i l l be at the expense of the large, slow-growing females, the MS females. As a r e s u l t , FM phenotypes with i n t e r -mediate growth w i l l predominate i f a population i s heavily f i s h e d . Thus, one i n t e r p r e t a t i o n of the decrease i n MS genotypes and the increase i n FM genotypes i s that a m u l t i - a l l e l e "Ricker" e f f e c t has taken place i n the Pegasus Bay t a r a k i h i population as a r e s u l t of i n h e r i t e d differences i n growth rate associated with a l l e l i c genotypes at the PGM locus. However, there are other possible explanations. Gauldie and Johnston (1980) suggest that there may be a general north-south c l i n e i n PGM a l l e l e frequency f o r Cheilodactylus macropterus cor r e l a t e d with temperature. Temperature differences of about 3.5°C occur between East Cape and B l u f f . S i m i l a r temperature differences at the time of spawning 113 i n 1966 and 1973 may have caused a difference i n a l l e l e frequency of those year classes s i m i l a r to that described by Smith (1979) f o r the esterase locus i n the snapper, Chrysophrys auratus. The r e l a t i o n s h i p between growth, a l l e l i c phenotype, and f i s h i n g pressure may not be so r e a d i l y demonstrated f o r Other enzymes and other species. But i t i s reasonable to expect that electrophoretic studies, which pursue the i d e n t i f i c a t i o n of "stocks" by using small samples and assume that a l l e l e s are neutral markers of the genome, run the r i s k of l o s i n g valuable information, information that i s immediately relevant to the conservation and wise u t i l i s a t i o n of f i s h e r i e s . 114 REFERENCES G a l l u c c i , V.F. and T.J. Quinn 1979. Reparameterizing, f i t t i n g , and te s t i n g a simple growth model. Transactions of the American F i s h e r i e s  Society 108: 14-23. Gauldie, R.W. and A.J. Johnston 1980. The geographical d i s t r i b u t i o n of phosphoglucomutase and glucose phosphate isomerase a l l e l e s of some New Zealand f i s h e s . Comparative Biochemistry and Physiology 66(B): 171-183. Ray, W.J. and G.A. R o s c e l l i 1964. The phosphoglucomutase pathway. Journal of B i o l o g i c a l Chemistry 239(11): 3935-3954. Ricker, W.E. 1973. Two mechanisms that make i t impossible to maintain peak-period y i e l d s from stocks of P a c i f i c salmon and other f i s h e s . Journal of the F i s h e r i e s Research Board of Canada 30: 1275-1286. Smith, P.J. 1979. Esterase gene frequencies and temperature r e l a t i o n s h i p s i n the New Zealand snapper, Chrysophrys auratus (Forster). Marine Biology  37: 111-114. Stanbury, J.B., J.B. Wyngaarden and D.S. Fredrickson 1978. The metabolic basis of i n h e r i t e d diseases. McGraw-Hill, New York. 115 Tong, L.J. and CM. Vooren 1972. The biology of the New Zealand t a r a k i h i , Cheilodactylus macropterus (Bloch and Schneider). F i s h e r i e s Research B u l l e t i n No. 6> F i s h e r i e s Research D i v i s i o n . New Zealand M i n i s t r y of A g r i c u l t u r e and F i s h e r i e s . Vooren, CM. 1973. The population dynamics of the New Zealand t a r a k i h i , Cheilodactylus macropterus (Bloch and Schneider), and changes due to f i s h i n g : an exploration. Oceanography of the South P a c i f i c 19 72, comp. R. Fraser. New Zealand National Commission of UNESCO, Wellington. ACKNOWLB DGEMENTS We are greatly indebted to Professors Judy Myers, Don McPhail and Peter Larkin of the University of B r i t i s h Columbia, as well as to the unknown reviewers f o r t h e i r comments and advice. 117 THE ROLE OF ALLELIC VARIATION IN THE MANAGEMENT OF FISHES IV. Ion Content Relationships of Chinnok Salmon O t o l i t h s I Progress Towards Translating the E c o l o g i c a l Record of the O t o l i t h t R.W. Gauldie *J.R. Davidson and Janet P el F i s h e r i e s Research D i v i s i o n P 0 Box 297 Wellington, New Zealand New Zealand Research Advisory Council Fellow at The I n s t i t u t e of Animal Resource Ecology The University of B r i t i s h Columbia •*EPS-FMS Laboratory Services Environment Canada 4195 Marine Drive, West Vancouver B r i t i s h Columbia, Canada V7V IN8 118 ABSTRACT S i b l i n g chinook salmon (Oncorhynchus tshawytscha) were reared i n groups of 100 at 6 d i f f e r e n t average temperatures, 6.9, 8, 9.9, 11.7, 13.5 and 15.1°C f o r 43 days. The r e l a t i o n s h i p of rearing temperature to the ion content of both ara g o n i t i c and v a t e r i t i c o t o l i t h s i s examined f o r the ions Cd, Cu, Ba, Fe, Mg, Mn, Zn, P, Na and Sr. The consequences of t h i s r e l a t i o n s h i p f o r the p o t e n t i a l use of the o t o l i t h as an environment recording structure are discussed. 119 INTRODUCTION Va r i a t i o n i n the ion content of the o t o l i t h s of fishes has been reported f o r a number of species. Explanations of the differences i n o t o l i t h ion concentration between f i s h species and between groups of fis h e s within the same species have consideredlan unstated l o c a t i o n e f f e c t (MacPherson and Manriquez, 1977) , a l o c a t i o n e f f e c t due to temperature (Gauldie e t a l . , 1977, 1978, 1980) and a lo c a t i o n e f f e c t due to p o l l u t i o n (Papadopoulou et a l . , 1976, 1980). O t o l i t h s are widely c o l l e c t e d f o r f i s h e r i e s management purposes, p a r t i c u l a r l y f o r estimating the age of f i s h , and can be stored without loss of ions. The techniques a v a i l a b l e . f o r ion analysis are r e l a t i v e l y inexpensive and permit large number of o t o l i t h s to be assayed quickly. Thus the ion content of o t o l i t h s o f f e r s f i s h e r i e s managers a r e a d i l y accessible t o o l to explore stock structure, temperature l i f e h i s t o r y , or p o l l u t i o n e f f e c t s depending on which i n t e r p r e t a t i o n of the v a r i a t i o n i n ion content i s accepted. This paper describes an experiment aimed at t e s t i n g the e f f e c t of temperature on o t o l i t h ion content when d i e t and water q u a l i t y are cont r o l l e d . 12Q MATERIALS AND METHODS The f i s h used i n the experiment were drawn from a s i n g l e p a i r mating of Chinook salmon (Oncorhynchus tshawytscha) from w i l d stocks of the Quinsam River on Vancouver Island. Genetic control was approximated by r e s t r i c t i n g the experiment to the o f f s p r i n g of a s i n g l e p a i r mating. The f e r t i l i s a t i o n of eggs took p l a c e . i n October 1978. A l l the f i s h were subsequently reared together and fed to r e p l e t i o n on the same batch of Oregon Moist P e l l e t . M o r t a l i t y past the emergent f r y stage was very low, less than half-a-percent t o t a l m o r t a l i t i e s . On 20 August 1979, a random sub-sample of 500 f i s h were randomly sorted into 10 groups of 50 f i s h . These f i s h were then gradually acclimatised over 5 days to f i v e d i f f e r e n t temperature regimes each cons i s t i n g of two l o t s of 50 f i s h i n separate tanks. F a c i l i t i e s were provided by the P a c i f i c B i o l o g i c a l Station,. Nanaimo. For -the duration of o the experiment, temperatures were maintained as close as possible to 8 , 10°, 12°, 14° and 16° Celsius. However, the i n i t i a l acclimation period (of up to 5 days f o r the highest temperature regimes) leads to a lower mean temperature and increased standard error . The consequent mean d a i l y temperatures f o r each of the f i v e treatment groups over the 43 day experi-mental period were as follows (+_ 2s.e.)6.9 +_ - 1 ( c o n t r o l ) , 8.0+.004',. 9.9+.012, 11.7+.034, 13.5+.074,. 15.11+.17. During the temperature experi-ment the f i s h were fed to r e p l e t i o n on the same batch of Oregon Moist P e l l e t upon which they had been previously fed. A f t e r 43 days a l l of the f i s h were anaesthetised, weighed, measured, frozen i n l i q u i d nitrogen, and stored at -20°C. 121 v. During the 43 day temperature experiment 14 f i s h died i n the lowest temperature tanks. Examination of these f i s h f a i l e d to reveal any parasites or i n f e c t i o u s agents. At the end of the experiment a further sample of 100 f i s h was taken randomly from t h e . o r i g i n a l population. They were anaesthet-ised, weighed, measured, and frozen i n l i q u i d nitrogen and stored at -20°C. When the o t o l i t h s were removed-it was evident that two morphological types of o t o l i t h s were present, an ara g o n i t i c , or normal o t o l i t h , and a v a t e r i t i c type. Aragonite.and v a t e r i t e are two c r y s t a l l i n e forms of calcium carbonate. A l l the o t o l i t h s were scored on a scale of 1 to 5, ranging from normal aragonitic (1)., through apparently mixed ara g o n i t i c and v a t e r i t i c types (2-4), to v a t e r i t i c only types (5). The s c a l i n g i s demonstrated i n Fig.. 1. Upon removal, r i g h t and l e f t o t o l i t h s were kept separate and at that time the f i s h were sexed. A l l the l e f t o t o l i t h s and a sub-sample of the r i g h t o t o l i t h s were then analysed by emission spectrography f o r the ions cadmium (Cd), copper (Cu) , I r o n (Fe), magnesium (Mg), manganese (Mn), zinc (Zn), barium (Ba), phosphorus (P), sodium (Na), and strontium (Sr). Each o t o l i t h was weighed i n a 10 ml acid-washed beaker, then exposed to an oxygen bearing plasma i n a low temperature asher (International Plasma PM-448) to decompose the organic material. The remaining inorganic material was dissolv e d with 0.05 ml of concentrated n i t r i c a c i d (Aristar) and d i l u t e d by adding 3.5 ml of a so l u t i o n containing 11.43 mg/1 holmium. The f i n a l volume was 4.0 ml and the f i n a l holmium concentration was 10 mg/1. The sample s o l u t i o n was then transferred to an acid-washed polystyrene tube and capped. 122 F i g . 1 The range of mixed a r a g o n i t i c / v a t e r i t i c Chinook salmon o t o l i t h s from (1) most aragonitic to (5) most v a t e r i t i c . A l l o t o l i t h s are viewed from the i n t e r n a l , anti-sulcus side. (1) 123 F i g . 1 continued (5) 125 The samples were analysed by Inductively Coupled Agron Plasma O p t i c a l Emission Spectrometry ( J a r r e l l - A s h 975 Atocomp with spectrum s h i f t e r ) . Standards contained the 1Q determined elements Cd, Cu, Fe, Mg, Mn, Zn, Ba, P, Na, Sr, the i n t e r n a l standard holmium, 12.5% n i t r i c a c i d and 1QQ mg/l of calcium to matrix-match the sample. Detection l e v e l s were s e t a t 10 times the standard deviations of blanks. The usual lowest quantity determinable with t h i s technique i s 5 times the standard deviation of blanks (Butler et a l . , 1975), but the v i s c o s i t y of our samples was high and v a r i a b l e since they had calcium concentrations ranging from 800 to 1200 mg/l. To overcome anomalies generated by flow rate differences due to d i f f e r e n t v i s c o s i t i e s the lowest quantity determin-able was set at 10 times the standard deviation of blanks. Before any s t a t i s t i c a l analysis can be c a r r i e d out i t i s necessary to e s t a b l i s h the r e l i a b i l i t y of the data. A measure of t h i s can be made by d i v i d i n g the lowest quantity determinable (10 standard deviations of the blanks) by the minimum difference between the mean values of temperature treatment groups f o r each ion. Expressed as a percentage t h i s 'minimum meaningful difference" f o r each ion i s Fe, 2.2%; Mg, 3Q%; Mn, 52.5%; Zn, 0.8%; P, 2.2%; Na, 0.9% and Sr, 0.02%. Ov e r a l l , o t o l i t h s i z e increases with f i s h s i z e , but within temperature treatment groups there was a considerable v a r i a t i o n i n s i z e of f i s h and o t o l i t h s , so the ion content of o t o l i t h s was standardised to o t o l i t h weight. A l l s t a t i s t i c a l analyses were c a r r i e d out on the i o n / o t o l i t h weight r a t i o expressed as ppm/mg. Approximate values of the mean ion content" of o t o l i t h s for comparative purposes were obtained by multi p l y i n g the mean i o n / o t o l i t h weight for the 8°C temperature treatment group by the mean o t o l i t h weight of that group (Table 3). 126' The frequency d i s t r i b u t i o n of the i o n / o t o l i t h weight r a t i o i s skewed to the r i g h t hand side. These frequency d i s t r i b u t i o n s were not normalised by a logarithmic transformation, and consequently a non-parametric analysis of variance, the Kruskal-Wallis t e s t , was used to determine the s t a t i s t i c a l s i g n i f i c a n c e of differences i n i o n / o t o l i t h weight r a t i o among temperature treatment groups. RESULTS Two morphological types of o t o l i t h were recovered. These were the normal aragonitic type and an abnormal v a t e r i t i c type that consists of a mixture of v a t e r i t e and aragonite (Fig. 1). V a t e r i t e i s an a l t e r n a t i v e c r y s t a l l i n e form of calcium carbonate. Both types of o t o l i t h occur together i n some f i s h e s , one on each side of the head. The proportion of occurrence of i n d i v i d u a l f i s h with.both o t o l i t h s a r a g o n i t i c was 74.5%, and with both o t o l i t h s v a t e r i t i c i t was 4.5%. In the mixed v a t e r i t i c / a r a g o n i t i c i n d i v i d u a l s (21%), the v a t e r i t i c types occur on the l e f t and r i g h t hand side i n approximately equal proportions. The ions Cu, Cd, and Ba r a r e l y exceeded t h e i r detection l i m i t s . With so few detectable values a v a i l a b l e they were discarded from t h i s study. A l l the i o n / o t o l i t h r a t i o s except v a t e r i t i c Fe showed a s i g n i f i c a n t heterogeneity of means (Tables 1, 2) i n both ar a g o n i t i c and v a t e r i t i c o t o l i t h s . The v a t e r i t i c Fe sample sizes were very small (5) which may account f o r t h i s r e s u l t . The e f f e c t of treatment temperature on the ion concentration of the v a t e r i t i c o t o l i t h s i s shown i n F i g . 3. The Fe l e v e l s i n the v a t e r i t i c o t o l i t h s are very low so that there were very few observations with Fe values i n excess of the detection l i m i t . Generally, Mg l e v e l s i n the v a t e r i t i c o t o l i t h s were almost 10 times higher than i n the ar a g o n i t i c . In general, the ion concentrations of P, Fe, Zn and Mn i n v a t e r i t i c o t o l i t h s d i d not d i f f e r appreciably from aragonitic o t o l i t h s , but Sr and Na values were lower. The v a t e r i t i c o t o l i t h s covered a range of mixtures of aragonite and v a t e r i t e (Fig. 1), so that the pattern of response to treatment temp-erature i s d i f f i c u l t to i n t e r p r e t . The s t a t i s t i c a l t e s t f o r heterogeneity of means amongst the v a t e r i t i c temperature treatment groups are no longer appropriate since i t i s already known that they are pooled from a range of o t o l i t h types. (The Kruskal-Wallis t e s t does i n f a c t indicate a s i g n i f i -cant heterogeneity of temperature treatment means for a l l ions except Fe for v a t e r i t i c o t o l i t h s . ) However, the r e s u l t s do show that a s h i f t i n the c r y s t a l l i n e , form of the o t o l i t h causes a substantial change i n the r e l a t i v e amounts of some of the minor ion components of the o t o l i t h , strongly suggesting that these ions are an i n t e g r a l part of the normal c r y s t a l forming process. Temperature treatment group mean ion concentrations and standard errors f o r a r a g o n i t i c o t o l i t h s expressed as average ion (ppm) per weight of o t o l i t h s (mg). Temperature 6.8 1.87 000.54-006.70 20 5.01 002.50-026.20 48 .51 000.32-000.74 84 13.1 007.60-152.00 85 52.06 015.50-245.00 41 451 242.00-732.00 85 82 039.00-110.00 85 8.0 2.82 000.76-0018.10 11 5.63 002.50-0031.90 25 .51 000.28-0001.39 79 10.01 007..00-0020.00 64 38.22 019.60-0121.00 27 462 210.00-2198.00 64 73 027.00-0099.00 63 9.9 4,4 000.59-012.1 10 .49 000.23-002.3 62 .9.9 -007.30-020.1 79 34.26 017.40-076.1 74 389 198.00-561.0 76 75 014.00-098.0 79 11.7 3.39 000.50-017.4 19 4.99. 002.50-042.5 64 .55 000.31-001.4 76 12.4 007.70-071.4 76 44.6 030.40-058.7 8 515 309.00-807.0 76 92 043.00-119.0 76 2.19 000.60-007.00 17 4.67 002.50-036.70 44 .57 000.36-000.98 60 12.33 008.20-018.70 60 45.8 019.20-086.80 33 542 264.00-811.00 60 97 029.00-120.00 60 1.55 000.50-005.90 20 5.10 005.10-027.10 4 .58 000.20-000.93 75 13.13 008.10-019.20 75 46.2 022.30-112.00 74 427 228.00-625.00 98 034.00-128.00 75 M ' J to co Table 2. Temperature treatment group mean ion concentrations and standard errors f o r v a t e r i t i c o t o l i t h s expressed as average ion (ppm) per weight of o t o l i t h (mg). Ion Temperature 6.8 8.0 9.9 11.7 13.5 15.1 Fe range N 2. 001.20-5 09 003.13 2. 000.94-3 92 006.60 2.26 001.20-005.00 4 1.08 1.79 000.90-001.3 001.20-002.81 3 4 Mg 34.5 30.9 20.6 range 006.60-048.60 007.20-048.20: 016.50-027.90 N 22 10 6 25.07 003.70-043.10 19 41.29 36.65 008.80-059.2 019.00-068.70 19 17 Mn range. N .78 .74 .61 000.40-001.20 000.37-001,13 000.28-000.91 24 14 20 .72 .85 000.45-001.01 000.43-001.33 19 22 .82 000.29-001.23 24 Zn range N 12.45 12.48 10.45 008.10-017.90 007.60-022.60 006.60-019.90 24 14 20 11.83 13.62 008.30-018.00 009.80-023.40 19 22 14.18 006.90-028.60 24 P 50.5 37 range 024.00-076.00 023.00-055.00 N 9 " 5 28 014.00-045.00 18 46 031.00-064.00 4 45 023.00-078.00 12 46 019.60-088.80 20 Na 303 311 range 198.00-542.00 189.00-N 24 14 290 434.00 198.00-508.00 20 447 385 299.00-635.00 230.00-19 22 353 636.00 236.00-526.00 24 Sr 39.3 44.3 46.9 range 021.50-072.80 013.50-073.70 017.70-081.10 N 24 14 20 61.9 027.70-115.00 19 55. 026.90-22 8 098.40 59.4 >0-] 24 026.60 1079.0 £ KD 130/ DISCUSSION Mugiya (1975) i d e n t i f i e d a v a t e r i t i c c r y s t a l l i n e o t o l i t h i n Salmo  gairdneri which can be seen from his photograph to be very s i m i l a r to the v a t e r i t i c type described i n t h i s study. The proportion of aragonitic and v a t e r i t i c types described '-in t h i s study do not r e f l e c t the proportion of v a t e r i t i c o t o l i t h s i n w i l d populations.of chinook, although the proportion of such o t o l i t h s has been reported to be quite high (20%) i n l o c a l popula-tions of other species (Johansson, 1979). The proportion of v a t e r i t i c o t o l i t h s does not change with temperature or with the weight or length of the f i s h . The proportions observed do not r e a d i l y lend themselves to a genetic explanation, but the scoring may not r e f l e c t the true d i s t r i b u t i o n of types. Careful examination of the other two o t o l i t h s i n the semi-circular canal, the astericus and l a p i l l u s , revealed no abnormalities even when an almost completely v a t e r i t i c s a g i t t a was present. One can draw two conclus-ions from t h i s observation. F i r s t , that the abnormality i s not induced by a disease organism since - i t does not a f f e c t an i d e n t i c a l process occurring a short distance away i n the same f l u i d medium. Second, by the same reasoning, the c r y s t a l deposition process cannot be a simple p r e c i p i t a t i o n out of the endolymphatic f l u i d ( I r i e e t a l . , 1967; Mugiya et a l . , 1979). Skewed d i s t r i b u t i o n s of ions l i k e those found i n the o t o l i t h s examined are widely reported i n studies of various organs of fishes (Harms, 1974). The reasons f o r t h i s general skew are not c l e a r . The most widely accepted explanation i s that the instantaneous ion concentration of a tis s u e i s the net r e s u l t of a number of simultaneously changing k i n e t i c processes. The arguments supporting t h i s explanation are discussed i n 131 d e t a i l f o r f i s h e s by Giesy and Wiener (1977) and more generally f o r many organisms by Best and Hearon (1960). Giesy and Wiener (1977) argued that the most common tiss u e ion d i s t r i b u t i o n f i t s a log-normal curve better than any reasonable a l t e r n a t i v e curve. A logarithmic transformation did not normalise any of the o t o l i t h ion d i s t r i b u t i o n i n t h i s study, and consequently we can make two arguments f o r a r e l a t i o n s h i p between o t o l i t h ion content and temperature. The f i r s t i s that for aragonitic o t o l i t h s the ions Fe, Zn, P, Na, and Sr (those f o r which we have r e l i a b l e data) show a consistent pattern of mean ion content v a r i a t i o n with temperature with maxima or minima at 9.9°C (Fig. 2). The second argument i s that the differences i n mean ion content between temperature treatment groups are greater than could be expected from chance. A non-parametric analysis of variance, the Kruskal-Wallis t e s t , confirms that there i s a s t a t i s t i c a l l y s i g n i f i c a n t heterogeneity amongst temperature treatment group means including Mn and Mg. Mn and Mg do not show an 'acclimation' pattern with temperature treatment l i k e the other ions, but r e l i a b i l i t y of the data for these two ions i s low. The r e s u l t s of these experiments confirm the e a r l i e r work of Gauldie and Nathan (1977), Gauldie et a l . (1978), and Gauldie et a l . (1980) that o t o l i t h ion concentrations, p a r t i c u l a r l y Fe, are r e l a t e d to ambient temp-eratures. Gauldie et a l . (1980) reported a minimum and maximum for Fe of 17.75 to 34.45 ppm f o r the temperature range 6.8°C to 9.9°C f o r the i n t r o -duced Chinook salmon i n New Zealand. This compares well with the minimum and maximum, 19.14 and 45.04 ppm, over the same temperature range (6.8°C to 9.9°C) reported here. Comparisons with other reported values i n the 13 2 F i g 2. The mean metal ion content f o r each aragonitic temperature t r e a t -ment group p l o t t e d against mean treatment temperature. 133 13.4 l i t e r a t u r e f o r many, species in d i c a t e that our r e s u l t s are consistent with generally observed l e v e l s of ions (Table 3). Other reports of ion concentration being a function of p o l l u t i o n (Papadoupoulou e t a l . 1976, 1980) used very small samples . (12). Given the non-normal d i s t r i b u t i o n of Fe and Zn, the data i s d i f f i c u l t to compare with the present study. But the decline i n o t o l i t h ions with increasing age reported by Papadoupoulou e t a l . (1980) could be due to the normal movement of older, larger f i s h to deeper and colder water. Before the e f f e c t of temperature on o t o l i t h ion content can be put to any p r a c t i c a l use some problems must be overcome. The f i r s t of these i s recovering information about the ion content of o t o l i t h s . Can an o t o l i t h be prepared i n such a way that the ion l e v e l s can be read o f f the annuli (the average ion amount per annulus) using electron back-scattering techniques i n the way that Casselman (1974) has done f o r the calcium i n the annuli of the pike cleithrum? The need f o r t h i s step can be seen by reducing the treatment temperas ture to the average l i f e t i m e temperature, i . e . , the t o t a l degree-days divided by the t o t a l days instead of ju s t the 43 day treatment period. When t h i s i s done the 'treatment' group average temperatures 6.9, 8.0, 9.9, 11.7, 13.5, 15.11 go down to 8.36, 8.47, 8.71, 8.93, 9.16 and 9.35°C re s p e c t i v e l y . Yet i t i s obvious from the nature of the experiment that the differences i n ion concentrations were generated by the temperature t r e a t -ments. The change i n o t o l i t h s i z e with temperature treatment i s d i f f i c u l t to obtain since the o t o l i t h weight i s not highly correlated with f i s h weight (Table 3), and shows i n f a c t a v a r i a b l e response to temperature treatment, the f i s h a t the upper and lower temperature, treatment l e v e l s showing the l e a s t c o r r e l a t i o n between o t o l i t h weight and f i s h weight. 135 F i g . 3 The mean metal ion content f o r each v a t e r i t i c temperature treatment group.plotted against mean treatment temperature. 136 Table 3: Ion composition of o t o l i t h s percent: Sr Na ppm Mg Zn Mn Fe aragonitic Oncorhynchus tshawytscha , (1) v a t e r i t i c Oncorhynchus. tshawytscha (1) Gadus (sp) (2) Oncorhynchus tshawytscha (3) Oncorhynchus nerka (3) Merluccius capensis. (4) Scomber japonicus colas (5) .070 .39 .045 .31 .300 .33 .014 .32 40 316 400 20 846 379 310 103 127 5 8 54 30 29 20 23 15 4-10 The data are drawn from the following: (1) average values from the 8°C temperature treatment group of t h i s study; (2) Milliman (1974); (3) Gauldie, Graynoth and Illingworth (1980); (4) McPherson and Manriquez (1977); (5) Papadoupoulou et al.(1980) OJ 138 At present electron back-scatter measurements require a l a r g e r concen-t r a t i o n of ions than the low i o n l e v e l s l i k e l y to be found at the surface of a prepared o t o l i t h can be expected to provide. However, John Calaprice of the Scripps I n s t i t u t i o n of Oceanography has made progress towards the use of proton e x c i t a t i o n emission spectrography and proton beam ion sputtering techniques i n reading the ion content of the annuli i n f i s h spines. As t h i s technique i s further developed the problem of t r a n s l a t i n g the ion content at the o t o l i t h s annulus l e v e l w i l l be overcome. Thus at present one i s r e s t r i c t e d to whole o t o l i t h measurements which give r i s e to two problems. The f i r s t problem l i e s i n the concave and convex shapes of the ion/temperature curve. The i o n i c composition at both ends of the temperature scale i s s i m i l a r . However, the temperature t r e a t -ment range, about 8°C, i s quite large. Most commercial demersal marine f i s h species l i v e within an average temperature range of about 2.5°C. Consequently, a general knowledge of the biology of the f i s h would locate the end of the temperature range to which ion l e v e l s belong. The second problem i s much less t r a c t a b l e . None of the ions are normally d i s t r i b u t e d . An observation of the ion content of a p a r t i c u l a r o t o l i t h can thus place i t i n the modal range of one temperature or i n the t a i l of a temperature l e v e l above i t . Unfortunately, i t i s not p o s s i b l e to combine the observations of o t o l i t h ion concentrations i n t o some sort.o f m u l t i v a r i a t e s t a t i s t i c or discriminant function. The r a t i o n a l use of such functions requires that both the frequency of ions, and covariance between ions, are homogeneous (Snedecor and Cochran-, 1979). The covariance between ion p a i r s i s quite heterogeneous within and between ions and temperature treatment groups (Table 5), and the frequency d i s t r i b u t i o n of ions with temperature treatment 139 groups i s not homogeneous, ranging from nearly normal to grossly non-normal. Some of the high covariances i n Table 5 may be explained. For example, sodium occurs i n the o t o l i t h as p i r s s o n i t e (Na^ Ca^ (CO-j)2) or s h o r t i t e (Na^ Ca 2 (00^)2). , .5 H^O) (Morris and Kittleman, 1967), so one might guess that the more highly reactive Sr p r e f e r e n t i a l l y forms the analogous Na c r y s t a l . However, both Na and Sr show a higher covariance with o t o l i t h weight than a l l the other ions except Mn (Table 4), thus introducing the p o s s i b i l i t y that Na and Sr. i on l e v e l s are driven by some process concerned with o t o l i t h , s i z e . That such a process e x i s t s can be seen from the co-variance of o t o l i t h weight and f i s h s i z e (Table 4). At the slowest and f a s t e s t growing temperatures the covariance i s lowest which may indic a t e a top and bottom l i m i t on the o t o l i t h rate of growth process beyond which o t o l i t h growth becomes more independent of f i s h growth. I t has been shown that Mn and Zn are involved i n the p h y s i o l o g i c a l processes of o t o l i t h formation i n mice .(Erway and Po r i c h i a , 1974) , but Mn also has a higher covariance with o t o l i t h weight than Zn, suggesting yet another p h y s i o l o g i c a l r o l e . Other high covariances, l i k e those between Fe and P and, Mg and P, have not been reported i n the l i t e r a t u r e . 1 A number of studies i n c a l c i t i c benthonic foramiriifera have shown Sr and Mg l e v e l s to be biogenic and that Mg l e v e l s show a strong dependency on ambient water temperatures (Blackmon and Todd, 1959). A number of physiological-processes may be involved i n the response of the o t o l i t h ion to temperature. These processes require i n v e s t i g a t i o n before the temperature p r e d i c t i n g properties of the o t o l i t h ions can be f u l l y exploited. 14Q Table 4: The r e l a t i o n s h i p within temperature treatment groups of f i s h 2 weight and o t o l i t h weight for aragonitic o t o l i t h s ; r i s the square of the c o r r e l a t i o n c o e f f i c i e n t . Treatment temperature (°C): 6.8 8.0 9.9 11.7 13.5 15.1 2 r f i s h w t / o t o l i t h wt: .427 .473 .56 .367 .349 .291 Table 5: Inter-dependence of ion concentrations i n aragonitic o t o l i t h s , following covariance with temperature treatment groups. Sample s i z e i s i n brackets 15.11°C 13.5°C 11.7°C 8°C 9.9°C 6.8°C Na/Sr Mn/Zn Fe/Sr Fe/Na Fe/Zn Fe/Mn P/Sr Mg/Sr Fe/Mg Mg/P Fe/P Mn/P .61 (74) .24 (75) .001 (20) .03 (20) .0001 (20) .10 (20) .02 (74) .96 (4) .29 (4) .09 (20) .05 (74) .41 (60) .26 (60) .04 (17) .0002 (17) .0007 (17) .017 (17) .03 (33) .52 (44) .14 (13) .03 (18) .28 (11) .016 (33) .31 (76) .16 (76) .17 (19) .054 (19) .21 (19) .004 (19) .09 (8) .32 (64) .0006 (14) .85 (8) .23 (8) .42 (63) .10 (62) .19 (10) .47 (11) .007 (11) .73 (11) .11 (26) .62 (24) .97 (5) .92 (9) .75 (4) .19 (27) .43 (79) .16 (79) .23 (10) .0001 (10) .014 (10) .08 (10) .013 (74) .37 (85) .004 (84) -6 10 .04 .06 .01 (9) .0009 (74) .38 .15 .32 .08 (20) (20) (20) .005 (20) .008 (41) (48) (8) (27) (8) .0006 (40) 142 Table 6: Regression of mean gene and genotype frequency on the mean Fe content of o t o l i t h s from the same sample area from Gauldie and Johnston (1980) and Gauldie and Nathan (1977). 'Fast' F a l l e l e 'Medium' M a l l e l e 1 Slow' S a l l e l e FF genotype FM genotype MM genotype MS genotype SS genotype FS genotype FF genotype areas A and B FM genotype areas A and B MM genotype areas A and B MS genotype areas A and B SS genotype areas A and B FS genotype areas A and B .20 .00006 .019 .0014 .172 .168 .008 .004 .157 .37 .76 .61 .003 .014 .57 slope .019 -.00005 -.00004 .0097 .027 -.046 .009 -.003 .006 .006 .033 -.054 .003 -.001 .006 in t e r c e p t .028 .784 .157 .0115 .034 .71 .026 .043 .0012 .006 .032 .72 .238 .025 -.003 143 One a p p l i c a t i o n of the o t o l i t h temperature/ion r e l a t i o n s h i p would be to investigate the stock structure of f i s h e s . Oceanic water masses have c h a r a c t e r i s t i c temperature p r o f i l e s , so one might ask (i) do stocks of the same species have d i f f e r e n t o t o l i t h , ion contents corresponding to the water mass i n which they are found?; and ( i i ) are there genetic, i . e . , a l l e l i c , differences between these stocks? Gauldie and Nathan (1977) report three s t a t i s t i c a l l y d i f f e r e n t groups of o t o l i t h s with respect to t h e i r Fe content f o r New Zealand t a r a k i h i , Cheilodactylus macropterus, that correspond to the d i f f e r e n t oceanic waters of New Zealand. However, they do not take i n t o account the non-normal d i s t r i b u t i o n , of the Fe data so that t h e i r r e s u l t s may represent an over-analysis of the general north to south change i n temperature i n New Zealand waters. Gauldie and Johnston (198Q) published data showing the a l l e l i c v a r i a t i o n at the PGM locus f o r Cheilodactylus macropterus i n New Zealand waters taken from stations close to those sampled i n the o t o l i t h study. The regression of gene and genotype frequencies at the PGM locus from table 2 i n Gauldie and Johnston (1980) against the appropriate o t o l i t h Fe 2 l e v e l s are shown i n Table 6. The c o e f f i c i e n t of determination (r ) f o r the regression of calculated a l l e l e frequency (1 to 3, Table 6) on o t o l i t h 2 Fe i s low. S i m i l a r l y the r value for. the regression of genotype frequency 2 on o t o l i t h Fe i s also low, but the r values vary from almost zero to about 0.20. Gauldie and Nathan (1977) claim a s t a t i s t i c a l l y s i g n i f i c a n t d i v i s i o n of the o t o l i t h Fe data into three regions. Their regions A and B are the 2 temperature extremes of the range of Cheilodactylus macropterus. The r values of the regression of genotype frequencies on o t o l i t h Fe from the 144 regions A and B only are very high (0.37 to 0.76: 10-15 Table 6) f o r the F a l l e l e containing genotypes and the MM genotype (0.61); and are very low (0.003 and 0.014) f o r the MS and SS genotypes. That i s , i f the values from the C regions are eliminated, the regression becomes s i g n i f i c a n t . One i s tempted to explain t h i s e f f e c t as s e l e c t i o n f o r the F a l l e l e by temperature s t r e s s . However, Gauldie and Gazey (1982) have argued that f i s h i n g pressure can change a l l e l e frequencies through dependence of growth rate on a l l e l i c genotype. The region C of Gauldie and Nathan (1977) contains the most heavily f i s h e d populations of Cheilodactylus macropterus, so that i t i s possible that there r e a l l y i s a much stronger c o r r e l a t i o n of gene and genotype frequency i n that region that i s being overridden by the e f f e c t s of the f i s h e r y . Isolated observations can often be brought together i n an i n t e r e s t i n g and p l a u s i b l e fashion, but without any means of v e r i f i c a t i o n . This i s not the case f o r o t o l i t h ion studies l i k e the one we have quoted. Most f i s h e r i e s laboratories have stores of o t o l i t h s , whose o r i g i n s are accurately known,,drawn from water masses with stable and well known.temperature regimes. In addition, many f i s h e r i e s laboratories have a l l e l i c and other information on the same'populations, from which the o t o l i t h s were c o l l e c t e d . Thus, f o r a r e l a t i v e l y . s m a l l expenditure the generality of the r e l a t i o n s h i p between the o t o l i t h ion content, electrophoretic genetic composition, and water mass temperature c h a r a c t e r i s t i c s can be tested. I f t h i s r e l a t i o n s h i p does indeed extend to other species and other circumstances, then we.lwill have av a i l a b l e a new and powerful management t o o l . 145 ACKNOWLE DGEMENTS The experiments leading to t h i s paper were made poss i b l e by the generous material and f i n a n c i a l assistance provided by Canada F i s h e r i e s and Oceans. We are p a r t i c u l a r l y g r a t e f u l to Mr. A.S. Wood, Dr. T. Mulligan, Dr. R. Bret t , Mrs. Rose Curti s and the tec h n i c a l s t a f f at the Rosewall Creek Hatchery. Valuable advice and c r i t i c i s m on the form and content of th i s paper came from Professors Judy Myers, Don McPhail and Peter Larkin of the University of B r i t i s h Columbia, and Dr. T. Mulligan of the P a c i f i c B i o l o g i c a l Station, Nanaimo. 146 REFERENCES Best, J.B. and J.Z. Hearon 1960. Thermodynamic P r i n c i p l e s and Concepts. In: Mineral Metabolism, An Advanced Treatise. Eds: Comar, C.L. and F. Bronner, Academic Press, New York and London. Volume 1, Part A, pp 224-232. Blackmon, P.D. and R. Todd 1959. Mineralogy of some foraminifera as r e l a t e d to t h e i r c l a s s i f i c a t i o n and ecology. Journal of Paleontology 33: 1-15. Butler, Constance C., R.N. K i n i s e l e y and V.A. Fassel 1975. Inductively coupled plasma-optical emission spectrometry: a p p l i c a t i o n to the determina-t i o n of a l l o y i n g and impurity elements i n low and high s t e e l s . A n a l y t i c a l  Chemistry 47(6) .- 825-829. Casselman, J.M. 1974. Analysis of hard tissue of pike, Esox l u c i u s L., with s p e c i a l reference to age and growth. P. 13-27. In: Bagenal, T.B. (Ed). The Ageing of F i s h . Proceedings of an International Symposium. Unwin Brothers Ltd., Old Woking. Erway, L.C. and N.A. P o r i c h i a 1974. Manganese, zinc and genes i n o t o l i t h development. In: Trace element metabolism i n Animals #2, Proc. of the second International Symposium. Ed: W.H. Hoekstra. pp. 692-695. 147 GauldieR.W. and Adrienne Nathan 1977. Iron content of o t o l i t h s of t a r a k i h i ( T e l e o s t i : C h e i l o d a c t y l i d a e ) . New Zealand Journal of Marine and  Freshwater Research I I ( 2 ) : 179-191. Gauldie, R.W., D. P u r n e l l and D.A. Robertson 1978. Some biochemical s i m i l a r i t i e s and differences between two jack mackerel species, Trachurus  d e c l i v i s and T. novaezelandiae. Comparative Biochemistry and Physiology  518(B) : 389-391. Gauldie, R.W., E.J. Graynoth and J . Illingworth 1980. The r e l a t i o n s h i p of the i r o n content of some f i s h o t o l i t h s to temperature. Comparative  Biochemistry and Physiology 66(A): 19-24. Gauldie, R.W. and W.J. Gazey 1982. The influence of s e l e c t i o n and the use of a l l e l e frequency data i n f i s h e r i e s management. I I I . D i f f e r e n t growth curve parameters associated with a l l e l i c phenotypes at the phospho-glucomutase locus i n the t a r a k i h i (Cheilodactylus macropterus; Cheilodactylidae: T e l e o s t i ) . Submitted to Transactions of the American  Fi s h e r i e s Society. Giesy, J.P., and J.G. Wiener 1977. Frequency d i s t r i b u t i o n s of trace metal concentrations i n f i v e freshwater f i s h e s . Transactions of the  American F i s h e r i e s Society 106(4): 393-403. Harms, U. 1974. The l e v e l s of heavy metals (Mn, Fe, Co, N i , Cu, Zn, Cd, Pb, Hg) i n f i s h from on-shore and off-shore waters of the German Bight. Counsel! Internationale pour 1'Exploration de l a Mer 1974/E:5 F i s h e r i e s Improvement Committee. 148 I r i e , T., T. Yokoyama and T. Yamada 1967. C a l c i f i c a t i o n of f i s h o t o l i t h s caused by food and water. B u l l e t i n of the Japanese Society f o r S c i e n t i f i c  F i s h e r i e s 33: 24-26. Johansson, G. 1966. Contribution to the biology of the dab (Limanda  limanda L.) i n Icelandic waters. R i t F i s k i d e i l d a r 4(3): 18-23. MacPherson, E. and M. Manriquez 1977. Varaciones de algunos elementos constituyentes del o t o l i t o y sus relaciones con e l crecimento de Merluccius  capensis. Investigacion Pesquera 41(2): 205-218. Milliman, J.D. 1974. Marine Carbonates, Part I. Springer-Verlag. New York. Mugiya, Y., H. Kawamura and S. Aratsu .1979. Carbonic anhydrase and o t o l i t h formation i n the rainbow trout, Salmo g a i r d n e r i : enzyme a c t i v i t y i n the sacculus and calcium uptake by the o t o l i t h in_ v i t r o . B u l l e t i n of the  Japanese Society f o r S c i e n t i f i c F i s h e r i e s 45: 879-882. Mugiya,:.Y. 1975. On aberrant sagittas of teleostean f i s h e s . Japanese  Journal of Ichthyology 19: 11-14. Morris, R.W. and L.R. Kittleman 1967. P i e z o - e l e c t r i c property of o t o l i t h s . Science 158: 368-370. 139 Papadoupoulou, C , CD. Kanias and E. Maraitopoulu-Kassimati 1980. Trace element content i n f i s h o t o l i t h s i n r e l a t i o n to age and s i z e . Marine  P o l l u t i o n B u l l e t i n 11(3): 68-71. Papadoupoulou, C. and E. Kassimati 1976. Zinc content i n o t o l i t h s of mackerel from the Aegean. Marine P o l l u t i o n B u l l e t i n 9(4): 106-108. Snedecor, G.W. and W.G. Cochran. 1967. S t a t i s t i c a l Methods. Sixth E d i t i o n . Iowa State University Press, Ames, Iowa. 15Q CONCLUSIONS The actual observation that a population g e n e t i c i s t makes with e l e c t r o -phoresis i s not the frequency of genes or genotypes. The observation that he makes i s i n the form of t h i s t r i a d : observation = genotype * . l o c a t i o n * constraint. A gene or genotype i s found i n an i n d i v i d u a l organism at a p a r t i c u l a r geographical l o c a t i o n ; unless i n d i v i d u a l s m a t e r i a l i s e out of thi n a i r there must be some constraint or reason f o r these i n d i v i d u a l s to be there. Thus an observation i s a complex of e n t i t i e s bound together i n some s p e c i f i c way. The neutral theory of a l l e l i c polymorphism claims that f o r p r o t e i n a l l e l e s the pa r t of the t r i a d (location * constraint) can be replaced by stochas t i c processes generated by accidents of mutation rate and those sampling errors caused by f l u c t u a t i n g population s i z e . Any dynamic process, f o r example, a motor car engine, can be modelled i n a t r i v i a l sense by a stoc h a s t i c process, but the neutral theory of a l l e l i c v a r i a t i o n does not o f f e r t h i s kind of t r i v i a l model. On the contrary, i t asserts that the true, underlying process of a l l e l i c v a r i a t i o n i s , i n i t s essence, stochastic. I t i s c l e a r from the arguments and evidence presented i n t h i s thesis that an important element i n the (location * constraint) part of the observation t r i a d i s to be found i n the i n t e r a c t i o n between the physio-l o g i c a l properties of a l l e l e s and the environment. Consequently stoc h a s t i c models of gene frequency d i s t r i b u t i o n s can be regarded as being at best mathematical analogues of the true, underlying processes that determine the observed a l l e l e frequency d i s t r i b u t i o n s , i n s p i t e of the claims of neutral t h e o r i s t s to the contrary. 151 The arguments and evidence presented i n t h i s thesis go further by showing that some e l e c t r o p h o r e t i c a l l y i d e n t i f i a b l e genes and genotypes are l i k e l y to be involved i n the population dynamic parameters/ l i k e growth, and disease resistance. However, even when sing l e locus e f f e c t s can be i s o l a t e d , the genetical processes that appear to the observer as changes i n the frequency of occurrence of gene, or genotype, over time and space are s t i l l complex. R e a l i s t i c models of such genetical processes would require an extensive knowledge of the physiology of the organism under study. Faced with the expense and d i f f i c u l t y of acquiring such physio-l o g i c a l l y competent models o f - g e n e t i c a l processes one might well turn to the simpleminded s t o c h a s t i c and s t a t i s t i c a l models as being at l e a s t q u a l i t a t i v e i n d i c a t o r s of the genetical processes of population genetics. Unfortunately, s t a t i s t i c a l approaches are, as I have argued i n t h i s t h e s i s , l i m i t e d to dealing with pseudo^taxonomic issues i n f i s h e r i e s biology, issues that deal with the recognition and c l a s s i f i c a t i o n of the patterns of a l l e l i c v a r i a t i o n . The neutral theory i t s e l f l i m i t s any studies of a l l e l i c variation; to the comparison of patterns. Even with t h i s r e s t r i c t i o n i t might be argued that, f i s h e r i e s management i n t e r e s t i n a l l e l i c v a r i a t i o n ;is l i m i t e d to the d e f i n i t i o n of g e n e t i c a l l y homogeneous ( i . e . , randomly mating) groups that can be r e a d i l y i d e n t i f i e d by pattern matching s t a t i s t i c s . The general .arguments i n the f i r s t paper of the thesis would make one suspicious of such an argument.' Patterns of a l l e l i c v a r i a t i o n are l i k e l y to change under both, normal environmental v a r i a t i o n and f i s h i n g pressure. The second and t h i r d papers dealing with t r a n s f e r r i n arid phosphoglucomutase variants are concrete examples supporting those suspicions. 152 The general idea that genetic homogeneity i s useful to f i s h e r i e s managers i s based on the assumption that f i s h e r i e s management str a t e g i e s do not require a d e t a i l e d knowledge of the biology of the organism, only the knowledge that b i o l o g i c a l l y d i f f e r e n t groups can be defined and thus modelled i n i s o l a t i o n . For example,. stock and r e c r u i t r e l a t i o n s h i p s are by d e f i n i t i o n based on the.idea that the "stock 1 i s homogeneous so that the v a r i a t i o n i n 'stocks' and ' r e c r u i t s ' can be adequately described by a mean and variance. Furthermore, many model parameters, for example catch per u n i t e f f o r t , are derived from the dynamics of the f i s h e r y and not n e c e s s a r i l y from the dynamics of the f i s h , as Ricker (1973) has shown. There has been a tendency i n f i s h e r i e s management modelling to play down the importance of the biology of the f i s h , but I think that i t can be argued that the most recent modelling s t r a t e g i e s do i n f a c t require, more than ever before, a knowledge of the biology of the f i s h . My argument i s as follows. Quantitative models of the underlying processes involved i n the natural regulation of numbers of f i s h began with the work of Baranov between 1918 and 1925. I t was Baranov's i n t e n t i o n to provide the f i s h i n g industry with an explanation of the regulation of numbers i n f i s h as well*'as a s p e c i f i c theory that would make quantitative predictions of the e f f e c t s on f i s h i n g pressure. Baranov (1918) proposed a very simple natural law of regulation of numbers: the amount of a v a i l a b l e food determines the amount of f i s h . In reply to c r i t i c i s m s that t h i s view was too simple, Baranov (1925) c l e a r l y stated h i s p o s i t i o n : "And so I assume that the abundance of f i s h i n a basin i s determined p r i m a r i l y by the abundance of food and that a l l other natural and h i s t o r i c a l 153. factors are important mainly i n as much as they influence the amount of food." Baranov (1918) introduces two further assumptions that are nowhere stated c l e a r l y but do i n f a c t follow unavoidably from the use of the l o g i s t i c curve as an expression f o r growth of f i s h e s . These two c r i t i c a l assumptions are (1) that the system was i n equilibrium i n i t s natural state, and (2), most importantly, that there was an innate d r i v i n g force i n the ^vu system that would continuously s t r i v e , i n the face of a l l perturbations, to drive the system back to the 'natural' equilibrium l e v e l . This l a t t e r assumption i s fundamental to a l l subsequent management models, even though i t i s often unrecognised and introduced i n t o models v i a t h e i r mathematical technologies. The 'innate drive' assumption l i e s behind Baranov's argument that the rate of production of f i s h material or f i s h mass can be increased by f i s h i n g a c t i v i t y i f the f i s h i n g a c t i v i t y serves to remove the older, larger, l e s s e f f i c i e n t energy consumers. In t h i s way the f i s h e r y can be maintained at a point of maximum rate of production even though i t i s away from the 'natural' equilibrium point with respect to i t s age and s i z e structure. Baranov-'s approach to the t h e o r e t i c a l problem of adjusting the catch to maintain the rate of production at the desired l e v e l breaks down in t o three components. 1. There i s an i d e n t i f i a b l e law of regulation of animal numbers. 2. This law has two fundamental prop e r t i e s : a 'natural' e q u i l i b -rium value and an innate r e s t o r i n g force that w i l l return the system to t h i s 'natural' equilibrium value a f t e r perturbation, 3. A mathematical statement incorporating these fundamental properties presents the law of regulation of animal numbers i n a form that allows quantitative predictions of the optimum catch. 154 Since Baranov's e a r l y e f f o r t s there has been a large scale development of f i s h e r i e s management models. From t h i s development three general kinds of a t t i t u d e to modelling have emerged. The f i r s t of these attitudes follows, i n p r i n c i p l e , Baranov's approach. Models are seen as being p r i m a r i l y concerned with the natural processes of reg u l a t i o n and are biased towards the biology of f i s h , rather than f i s h e r i e s data. The second a t t i t u d e to modelling the b i o l o g i c a l law of regulation of numbers has been displaced by mathematical and s t a t i s t i c a l devices. T y p i c a l l y these models take some n components to the f i s h e r y and 'glue' them together with some s t a t i s t i c a l technique which usually has at i t s base a multiple regression. Thus the tendency i n Baranov's work to have the innate d r i v i n g force contained i n the mathematics i s now complete. The reasons f o r t h i s development are not d i f f i c u l t to understand. Fir s t ; , e c o l o g i s t s have had l i m i t e d success i n e s t a b l i s h i n g any law of regulation of numbers f o r any species (Krebs, 1978), and second, two of the fundamental properties of Baranov's system, an equilibrium value and an innate r e s t o r i n g force, are also properties of multiple regression. Opposition to these models stems l a r g e l y from the mathematical? problems associated with the assumption of equilibrium and the need to weight the multiple regression components to maintain - s t a b i l i t y i n the model. The r e s o l u t i o n of these d i f f i c u l t i e s has l e d e i t h e r back to the b i o l o g i c a l approach, the f i r s t model, with i t s decreased qu a n t i t a t i v e power, or to a t h i r d modelling strategy, adaptive management models. Adaptive management models recognise i n a frank and open manner the a r b i t r a r y nature of mathematical and s t a t i s t i c a l techniques as w e l l as the s i m p l i c i t y of the attendant assumptions. But, i n view of the f a i l u r e 155 of b i o l o g i s t s to develop a law of regulation of numbers f o r any f i s h species, proponents of these models claim that i t i s reasonable to t r e a t the behaviour of the f i s h e r y as i f i t were a mathematical, rather than a b i o l o g i c a l , problem. These models t r e a t the f i s h e r y as a data generating device whose mathematical behaviours can be regarded as analogues of the i n a c c e s s i b l e b i o l o g i c a l processes that drive the: device XWalters, 1975; Walters and Hilborn, 1976). We are now i n a p o s i t i o n where the simple s t a t i s t i c a l pattern-matching usage of protein a l l e l e s i s no longer adequate, even i f the neutral theory were correct.. F i s h e r i e s management models now require a d e t a i l e d knowledge of the biology of the f i s h , p a r t i c u l a r l y i n the area of growth and disease resistance, which, as I have shown i n t h i s t h e s i s , can be r e l a t e d to a l l e l i c genotype. Thus, p h y s i o l o g i c a l l y competent models of the genetical processes underlying population dynamics, processes that sometimes involve a l l e l i c phenotypes, are highlyC-desirable to f i s h e r i e s management s t r a t e g i s t s . The development of the o t o l i t h ion/temperature r e l a t i o n s h i p as a t o o l to measure part, at l e a s t , of an i n d i v i d u a l ' s l i f e h i s t o r y o f f e r s a means to avoid the expense and delay involved i n p h y s i o l o g i c a l studies. The tempera-ture l i f e h i s t o r y of an i n d i v i d u a l provides a d i r e c t , experimental measure of some of the information i n the (location * constraint) part of the observation t r i a d . We can ask where i n d i v i d u a l s have been and what were the genetic consequences, the 'constraints', imposed by those changes i n l o c a t i o n . . We can ask what does the f i s h e r y do to the machinery of the adjustment of a species to i t s predators and prey under d i f f e r e n t c l i m a t i c stresses. In short, we can return to "The question of the b i o l o g i c a l basis of f i s h e r i e s " . 156 CONCLUSION REFERENCES Ricker, W.E. 1973. Two mechanisms that make i t impossible to maintain peak period y i e l d s from stocks of P a c i f i c salmon and other f i s h e s . Journal of the F i s h e r i e s Research Board of Canada 30: 1275-1286.-Baranov, R.I. 1918. On the question of the b i o l o g i c a l basis of f i s h e r i e s . B u l l e t i n of the Bureau of F i s h e r i e s , Petrograd 1(1): 81-128. Baranov, R.I. 1925. Concerning the objections of Prof. N.M. Knipovich to the a r t i c l e "On the question of the dynamics of the f i s h i n g industry." B u l l e t i n of the Fishery Economy 8: 8-12. Krebs, C.J. 1978. Ecology: The experimental analysis of d i s t r i b u t i o n and abundance. 2nd e d i t i o n . Harper and Row, New York. Walters, C.J. 1975. Optimal harvest strategies f o r salmon i n r e l a t i o n to environment v a r i a b i l i t y and uncertain production parameters. Journal of the F i s h e r i e s Research Board of Canada-32: 1777-1784. Walters, C.J. and R. H i l l b o r n . 1976. Adaptive control of f i s h i n g systems. Journal of the F i s h e r i e s Research Board,of Canada 33: 145-159. 

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