BIOCHEMICAL GENETICS OF BROWN .(Salnio trutta L.)

Isozyme variation as an indication of natural population structures and genetic variation in

by John N. King, B.Sc.

M. Phil.

University of Edinburgh IWO

1w CONTENTS - Page

Declaration

Acknowl edgements

Abstract

SECTION 1 INTRODUCTION 1 1.1 THE TROUT 3

1.1.1 Trout of Rivers and Seas 5

1.1.2 Trout of Lakes 8

1.1.3 Phenotypic Plasticity of Trout 9

1.2 INHERITED DIFFERENCES 11

1.2.1 Stock Concept 12

1.2.2 Questioning Phenotypic Relationships 13 1.3 ELECTROPHORESIS 15

1.3.1 Isozymes 15

1.3.2 Genetic Interpretations 17.

1.4 ISOZYME RESEARCH IN SALMONIDS 19

1.4.1 Research on Pacific and Trout 20

1.4.2 Research on Atlantic of 26

1.5 LIMITATIONS AND OBJECTIVES 28

1.5.1 Objectives 29 SECTION 2 MATERIALS AND METHODS 31 2.1 MATERIALS 36 2.2 METHODS 37 2.2.1 Buffer Systems 41 2.2.2 Staining Systems 42

2.2.3 Nomenclature Systems 44

SECTION 3 RESULTS 46 3.1 LACTATE DEHYDROGENASE 47

3.1.1 LDH-5 53 3.1.2 LDH-B 55 3.1.3 LDH-A 58 3.2.1 Aiphaglycerophosphate Dehydrogenase 63

3.2.2 Creatine Phosphokinase 67 3.3.1 Malate Dehydrogenase 72

3.3.2 Malic Enzyme 75 3.3.3 Phosphoglucoisomerase 77 3.3.4 Phosphoglucomutase 78

3.4 OTHER LOCI EXAMINED 79

SECTION 4 DISCUSSION AND CONCLUSIONS 83

4.1 ISOZYME VARIATION IN SCOTTISH 84 4.2 POPULATION STRUCTURES 86 4.3 SYSTEMATICS 89

4.3.1 Interspecies Systematics 92

4.4 MAN'S INFLUENCE ON FISH STOCKS 95 4.5 CONCLUSIONS 99 Bibliography 103

Appendix 1 Glossary 118

Appendix 2 Statistical Analysis 121

TABLES AND FIGURES

Table 1 Average Heterozygosity Figures 23

Table 2 Staining 43

Table 3 LDH-5 Allelic Frequencies 52

Table 4 Allelic Frequencies Lake Bunnersjoarna Sweden 60

Table 5 AGP- Genotypic Distribution 65

Table 6 CPK-1 Allelic Frequencies 70

Table 7 MDH-2 Allelic Frequencies 74

Figure 1 Materials 32

Figure 2 Methods 38

Figure 3 LDH- Genotypes 49

Figure 4 AGP- Genotypes 64

Figure 5 CPK- Genotypes 69

Figure 6 MDH- Genotypes 73

Figure 7 Malic Enzyme Genotypes 76

Figure 8 Other Isozymes 81 DEC LA RATION

This thesis has been composed by myself and is the result of my own work except where acknowledged. ACKNOWLEDGEMENTS

This study was undertaken during the tenure of a Commonwealth

Scholarship at the Department of Forestry and Natural Resources. I would-like to thank the people of the British Council and

Association of Commonwealth Universities for their support during

this time. I would also like to thank my supervisor Dr. D. H. Mills

for his help during this study.

Acknowledgements are due to:

Dr. John Thorpe for taking an interest in this study and

providing salmon from the Almondbank hatchery.

Dr. David Dunkley for providing smolt from the Department of

Agriculture and Fisheries trap on. the North Esk.

The Tweed commissioners and bailifs for allowing me access to

brown trout from the Tweed and the agents of the Scaliscro Estate,

Lewis, for the brown trout from there.

-EJ. Tony Child, Alan Archibald and David Thatcher for help with

techniques and procedures.

Drs. Fred Allendorf and Tom Cross for their time and information.

Many of the staff and students of the Department who helped me

gather material, take photographs, etc., especially John Burns, Reg

Noble, and Steve Barbour. I would also like to thank Peter Dracht

for his time in reading and criticising this thesis.

My wife Patty for her help and patience. ABSTRACT

• The many polytypic forms of brown trout raise questions about the nature of this diversity. Nineteenth century naturalists had many species and subspecies classified for this salmonid fish. It has since become apparent that environmental factors are important, but just how important, and how much the differences are due to genetic

and evolutionary divergence, is still a matter of speculation. To

effectively look at this problem some method of assessing genetic

variability and identifying genotypes is required.

This thesis looks at isozyme data as a measure of genotype

expression. It surveys twelve isozyme systems with over twenty

loci. The isozyme systems are described as to their functional

basis, the polymorphisms that have been noted, and the validity of

the polymorphisms as genetic .indicators. Allelic frequency

information is gathered for some of these isozyme loci. From data

collected at four sites in Scotland and information from current

literature, evidence is assessed about the genetic structure of

trout populations. Marked genetic heterogeneity has been noted in some Irish and

Swedish populations and there is evidence that some population

structuring may be based on trout phenotypes. The significance of

this heterogeneity is discussed. Not enough data may yet be

available to determine if this heterogeneity indicates evolutionary

divergence; it may be explained as random drift acting on small

isolated populations. The possibility of loss of genetic diversity

in hatchery stocks is discussed, as is the effectiveness of using

isozyme data as an indicator of gene diversity. 1 INTRODUCTION

The ever increasing human pressure on the natural resources of our planet has made us aware that these resources are not limitless and their environments not indestructable. Among vulnerable resources are the salmonid , including the brown trout (Salmo

trutta, Linnaeus). With better and faster transportation routes and more leisure time there is virtually no stream or loch remote enough

that it does not feel increasing pressure. Riparian owners

and managers wishing to enhance their resource are turning more to

stocking with hatchery fish, introducing exogenous stocks, and

generally applying a more intensified agricultural management

strategy.

Salmo trutta like many of its related salmon and trout species is

an economically valuable resource. Besides the thriving angling and

related tourist industry, it is an important part of the commercial

salmon fishery, is often only slightly less valued than

salmon or grilse on the commercial market. Part of the management

of this valuable natural resource should be an awareness of its

genetic resources. This requires some method of identifying

genotypes; a very difficult thing to do in a species that shows

quite a remarkable range of phenotypic variation (1.1). The

existence of many diverse forms of S. trutta has generated a great

deal of speculation as to whether these are environmental

manifestations of a highly plastic genotype, or a function of

genetic heterogeneity and evolutionary divergence within this

species. Despite the inconclusive nature of much of this

1 speculation (1.1 and 1.2) it remains not only a stimulating topic, but a concern of practical importance to effective trout management.

This thesis is intended to look at some of the applications of the techniques of population genetics, particularly using isozyme data as a direct measure of genotype expression (1.3). If this relationship can be verified it should prove a powerful tool for analysing population structures. Can the trout of Scottish waters be considered as a genetically homogeneous race? Are population structures based on life history and ecological differences or are populations based on geographic barriers? How fine are populations subdivided? What are the effects of maintaining fish stocks through hatchery programmes on the genetic diversity of trout populations?

How effective is isozyme data as an indicator of gene diversity?

Many of these questions are still a matter of speculation, but it is

intended to look at the evidence as it presently exists. A more

detailed outline of objectives is given in 1.5. 1.1 THE TROUT

Brown trout (Salmo trutta, L.) has a wide natural range covering all but the southern extremities of Europe, west Asia and high altitude waters of North Africa. Its indigenous and introduced distribution are described in detail by McCrimmon and Marshall

(1968) and McCrimmon, Marshall and Gots (1970).

The phenotypic variability shown by Saimo trutta and related species has created a great deal of confusion over the taxonomic and systematic relationships. Shaw in his "Systematic Natural History of Pisces" (1804) lists 64 species of the genus Salmo, and this of course excludes the fishes of the American Pacific coast. Gunther

(1866) lists 10 species of trout inhabiting the British Isles.

Campbell (1972) gives a detailed historic review of the classification of Salmo trutta. The current view of one polytypic species of trout was espoused by Day (1887) who believed the differences in markings and migration habits were primarily due to environmental influences. Day's hypothesis has been supported and extended by most writers since ( Regan, 1911; Nail, 1930; Trewevas,

1953).

Berg (1932) lists 6 geographical subspecies, Salmo trutta trutta in northern and western Europe and five others from the

Mediterranean eastwards. Typing by ecological forms is also common;

Salmo trutta trutta ( or marina ) - sea trout

Salmo trutta fario - river dwelling trout

Salmo trutta lacustris - lake dwelling trout.

3 But geographical subspecies and other formal methods of splitting a polytypic species have debateable usage because of the often arbitrary nature of the delineations (Mayr, 1963; Behnke, 1972).

One of the best candidates for subspecies designation or for very closely related sibling species is the carpione" of ,

Italy. (Numan, 1953; Behnke, 1972). Salmo trutta carpione or Salmo carpione is a small benthic trout that spawns twice yearly in the lake depths. It is hypothesized (Mayr, 1963) that a double post glacial invasion of the lake took place by two different forms of S. trutta, the original adopting this unusual spawning behaviour thus remaining reproductively isolated from the secondary invasion.

The debates over the status of S. trutta are understandable when

included as one polytypic species are;

- large silver sea trout of the Tweed which range up to

9 kg. (Nail, 1930),

- small Highland trout that fully mature at less than 8 cm. (Darling and Boyd, 1964),

- large lacustrine trout that have been recorded up to

39 kg. (Neresheimer, 1937 from Behnke, 1972),

- the distinctive silvery trout of Loch Leven,

- black finned trout of peaty mountain pools,

- the "" trout of Irish and Scottish lakes that

have distinctive red coloration and markings,

- the sonaghen" of Lough Neagh and Lough Melvin in

Ireland with a single row of vomerine teeth and dark fins.

These are examples of the more strikingly different types of trout,

but there is in general a great deal of variation in colour,

4 markings and size. The basic "trout" form however is fairly constant and most variation is size related. Indeed this basic shape and structure so well serves the trout niche that it has not evolved markedly between long seperated salmonid species and comparisons often rely on quite fine differences in morphological and anatomical characters (Behnke, 1971).

1.1.1 Trout of Rivers and Seas

Brown Trout: Trout can change their colour and shade to suit their habitat and markings often depend on diet (Frost and Brown, 1967).

Morphological characteristics such as vertebrae number, maxillae and

pyloric caecae, are heavily influenced by environment (Tanning,

1952; Garside, 1966).

Sea Trout: Earlier workers (Day and Regan) recognized two races of

sea trout in the British Isles, an east coast and western race. The western race (sewen) differs in having a longer head, larger fins, suboperculum projecting beyond the operculum and other morphological

features. Nall (1930) maintained that, as with many of the other

sea trout types found in Scottish waters, these anatomical comparisons were not consistent enough to differentiate "races". He did agree, however, that using scale growth, east and west coast

fish could be separated, each having different life history

characteristics. The sewen he felt had great similarities to

Norwegian fish while the east coast fish resembled Baltic sea trout.

Many of the numerous varieties and races of sea trout that exist in

popular usage can often be attributed to the enthusiasm of anglers

5 or their misidentification (e.g. "Bull" trout for second spawning

Atlantic salmon).

Russian work on sea trout of the White Sea (Yevsin, 1976; 1977) using morphological characteristics found no significant differences between seasonal forms (summer and autumn running) and only slight differences between fish in different river systems. These differences are attributed by Yevsin to selection for morphological traits that confer hydrodynamical adaption to the river system.

Indeed even the differences that did exist between seasonal forms

Yevsin sees as a teleological response to the river condition at the season of entry.

Although Nail (1930) felt that some of the sea trout "types" were not at all distinctive, he recognized a clear difference between sea trout and fresh water resident brown trout, and criticized Dahl's view (Dahl, 1933) that the distinction was not so clear. Dahl thought that in Norwegian waters at least, brown trout of up to 7 years might migrate to sea and be recruited into sea trout stocks.

He pointed out that in some rivers only males were present as fresh water resident trout, all females haying migrated as sea trout. In other rivers many males and females stayed and spawned but could be recruited into sea trout stocks at any stage. Nall's view was that some brown trout might indeed travel to salt water, usually estuarine waters (slob trout), but that these and other brown trout were quite distinct from "true" sea trout.

Pemberton (1976) found in North Argyll sea lochs that there was a

I great variability of trout from "brown to well silvered "sea trout" with many shades in between; moreover besides the spring smolt run a class of autumn recruits of brown trout appeared to move down from the burns. Campbell (1972) reported that the two ecological forms were difficult to categorize and the relationship between them complex. He suggested that in the Tweed they interbreed freely and like Dahl's Norwegian fish migrate in accordance with Ricker's

(1938) model for sockeye salmon:

The forces that bring about migration act more strongly on-faster growing and female fish.

After a certain size and with the onset of maturity, there is inhibition of migration, but this time more strongly on males.

Introductions of brown trout into North America and the Southern

Hemisphere tend to confirm the complex interrelationship of brown trout and sea trout. Plantings of established brown trout lines have also resulted in the development of anadromous runs (MacCrimmon and Marshall, 1968; Arrowsmith and Pentelow, 1965). In the

Kerguelen Islands, south Indian Ocean (E.P. Beall, Centre d'Hydobiologie INRA, St. Pee-Nivelle, France, pers. comm.) introductions of French brown trout have followed the patterns of other , southern hemisphere plantings. In detailed on-going studies it is felt that stock identification as to brown trout or sea trout is difficult if not impossible and they can most likely be considered as a single panmictic population.

7 1.1.2 Trout of Lakes

Lacustrine Trout: As with most trout, those living in lakes vary a great deal, though there are often distinctive differences between them and river dwelling fish. Usually those of large lakes are silvery with dark spots, they grow larger, mature later and sometimes biennially (Frost and Brown, 1967).

Ferox Trout: In some of the large mineral poor lochs of Scotland the majority of trout are small and short lived, rarely exceeding 30 cm. or 5 years in age (Campbell, 1971). Sympatric to these are a group of very large and long lived trout with rare individuals exceeding

90 cm. and 20 years (Campbell, 1979). Besides the obvious size differences, these large trout, known as "ferox', are described as being deep gold in colour and heavily spotted, extending often to isolated spot patches on the ventral surface (Campbell, 1979).

These markings and colour though are highly variable, Campbell

(1979) found ferox ranging from highly spotted individuals to females with only a few black spots on a silvery background that resembled fresh run salmon. Ferox are nearly always associated with charr ( alpinus, L.) and the common view is that they are long lived carnivorous individuals, that are able to exploit the charr shoals (Mills, 1971). Campbell (1979) lists three conditions that must be met for an individual to exploit this new niche.

1- It must live long enough and be late enough in maturing that it can reach a critical length of 35 cm. at between 6 to 7 years. This is the size at which it can effectively predate the charr shoals.

i 2- At this size it must switch its feeding from invertebates to fish and increases its growth rapidly. 3-. It must have the environmental conditions and potential to continue to live for a long. time.

The large nutrient poor lochs of Scotland provide this environment and Alm (1959) has shown that oligotrophic conditions favour long living, late maturing fish.

Gillaroo Trout: A trout found in Irish and Scottish lakes and noted

for numerous red spots above and below its lateral line; is said to

live only on molluscs and have a hard thick walled stomach. (Frost

and Brown, 1967). Its distinctive colouring and stomach thickness

is associated with its specialized diet.

1.1.3 Phenotypic Plasticity of Brown Trout

Most of the ecological work focusing on brown trout and its

phenotypes would tend to support Day's hypothesis that environmental

factors have a major if not overriding influence on migration

habits, markings, and size. Kosswig (1973) states that many of

these trout phenotypes are "merely modifications based on the wide

reaction norm of the genotype" and that pre-adaptions to ecological

specialization are "anchored in the genotype". Thus it could be

that most brown trout have the genetic programme to become a sea

trout; this seems to be borne out where introductions have been

carried out. Along this same line it is interesting to speculate

whether the large (ferox) are not just individuals lucky

enough to have the size advantage to push them into another ecological niche. In this new niche, as a piscivorous predator, it can overshoot the normal life and growth curves of its smaller brothers and sisters. Genetic differences can be important in this model; genotype Gl may have an easier trigger mechanism to the expression of phenotype P1 in environment El. Natural selection can act on these genetic differences. In the introduction of French freshwater brown trout to the Kerguelens, for example, natural selection would favour genotypes that are best able to express the migratory phenotype in this harsh environment.

10 1.2 INHERITED DIFFERENCES

Environmental factors undoubtedly play a major part in the formation of these trout phenotypes but inherited factors may also be important. The determination of genetic differences between natural stocks is difficult, comparing populations in the field does not remove the important environmental influences and controlled environment studies are obviously time consuming and expensive.

The most extensive work that investigates genetic differences between wild stocks of brown trout is that of Gunnar Alm (1949,

1959) at the Kalarne hatchery in Sweden where several generations of lacustris (lake trout) and fario (river trout) were kept in pond conditions. Many features including general coloration and fin colour persisted over the generations indicating that they were inherited. Skrochowska (1952) reared sea trout to maturity in ponds and found that a migratory instinct seemed to be maintained in the first generation but was lost in subsequent generations. In both of these studies growth rates appeared to be mainly influenced by the environment.

Ricker (1972) in his review of the effects of heredity and environment on Pacific salmon and trout states that earlier workers had underestimated genetic influences. In over twenty traits examined he concluded that differences in expression between stocks would be partly environmental and partly inherited.

11 1.2.1 Stock Concept

Ricker (1972) emphasized the stock concept and stressed that as both seasonal return and homing instinct are well developed, spawning isolation can maintain discrete populations. The results of the artificial selection work in plants and demonstrate the "widespread genetic variation for genes that are relevant to characters of adaptive significance" (Lewontin; 1974). Thus spawning isolation can maintain and strengthen genetic differences that arise through natural selection and genetic drift.

This unique "stock" concept can apply in comparing populations between river systems; but how finely populations are subdivided, and whether different phenotypic forms, life history forms, ecological or seasonal forms can be considered discrete stocks is difficult to assess. Ricker (1959) believed that "scores of

genetically different stocks of a species can exist in one and the

same river system and half a dozen or more within a single lake".

Ricker's belief was perhaps an exaggerated reaction to early workers

that had ignored the natural stock structures of the Pacific

salmonids, especially when making introductions and establishing

hatchery programmes.

To maintain discrete but sympatric stocks some spawning

segregation should occur either spatially or temporally. Stuart

(1957) found strong homing to individual spawning streams in brown

trout in a small Perthshire reservoir with only one noted stray from

3000 marked fish. This evidence of homing in a small lake does not

12 necessarily imply homing would be as exact to sea trout and migratory brown trout in larger water systems. In the much larger

Loon Lake of central British Columbia Northcote (1962) found some degree of straying between inlet and outlet spawning streams in .

Campbell (1979) conjectures that ferox might maintain genetic

integrity by selecting deeper water with large gravel redds and

inhabit large lochs with many big spawning streams. Gardner (1976)

points out that "spring-fish" are more likely to occur in larger

river systems with more and variable spawning grounds that allow a

chance of segregation between these Atlantic salmon types. Yevsin

(1976) felt that seasonal runs of sea trout from the White Sea chose

different stretches of the river to spawn. Campbell (1972) claims

that spawning between sea trout females and brown trout males might

be quite common in the Tweed. Jonsson and Sandlund (1979) claim

both dwarf and larger migrating male brown trout participate in

spawning in the same tributaries of a Norwegian river. Most of the

these paper's dealing with phenotypic segregation are only

speculative, though Campbell (1972) did claim to see the spawning

between brown trout and sea trout.

1.2.2 Questioning the Relationships Between Trout Forms

Trying to identify stock structures and the relationship between

them is a very difficult task. Campbell (1972), after his study of

brown trout and sea trout on the Tweed, could only conclude that the

relationship was complex and that it was "almost impossible to find

13 a criterion for phenotypic separation" between these two ecological forms. Because this variable species cannot be easily classified into discrete polymorphisms the questions we might seek to ask about these relationships are difficult to define.

The answers that have been forthcoming are inconclusive in many

ways. Lewontin (1974) has pointed out that the literature of

evolutionary genetics, as with population ecology, often sets

tolerance limits that are established strictly by conveniance, and

offers answers that cannot be empirically justified. To answer the

questions of evolutionary relationships Lewontin suggests that the

key is the description of genetic variation in populations; thus we

must look to the field of population genetics and the new techniques

of molecular biology that allow us to asses genetic variation in

natural populations. Once this initial variation has been described

it is possible to establish the systematic relationships between

populations.

14 1.3 ELECTROPHORESIS

The Watson-Crick (1953) model of the double helix DNA strand that provides the blue print for protein synthesis is the theoretical basis that allows us to use protein variations as a direct indication of genetic variation. Starch gel electrophoresis (Smithies, 1955) and histochemical staining (Hunter and Markert, 1957) have provided the techniques to indicate the variation.

Electrophoresis is based on the differential migration of water

soluble proteins through a support matrix (usually potato starch gel)

under direct current. Differences in the net charge of the protein molecules, and to a lesser extent size and configuration, will cause

the differential migration. Staining for general proteins of blood

and muscle have produced limited results, usually based on counting

and comparing banding patterns; this method has been used for species

and genus delineation (Nyman, 1967; Gardiner, 1974). Isozyme data,

which is based on the development of staining techniques that can

rapidly visualize genetically based sites of specific enzyme activity

on the gel matrix, has proved the real worth of electrophoresis in

looking at population structures.

1.3.1 Isozymes

An enzyme protein is composed of a set number of polypeptide

chains (subunits), these chains being coded by structural gene loci.

Monomeric enzymes consist of one subunit, dimers two, tetramers four

etc.. The final structure is known as the quaternary structure and

is constant for a specific enzyme. Isozymes (or isoenzymes) are multiple molecular forms of an enzyme that catalyses the same reaction but may differ in kinetic properties and charge due to differences in subunit make-up.

The polypeptide subunits that form the different isozymes originate from two basic sources (Harris and Hopkinson, 1976);

multiple gene loci that code for structurally distinct polypeptide chains of the enzyme,

multiple alleles at the same locus that code for distinct but equally valid polypeptide subunits.

These polypeptide chains coded from different loci or different alleles at the same locus, may differ due to point mutations that cause amino acid substitutions, which in turn can affect the net charge of the polypeptide subunit. These subunits joining up randomly will form a series of enzymes (isozymes) that differ in net charge.

Over the course of evolution with increasing biochemical complexity enzyme synthesis has been adjusted to the metabolic activities of particular tissues. Thus different isozymes of an enzyme are expressed in different tissues, also from different parts of a cell; some isozymes being found in the mitochondria, others in the extra-mitochondrial cytoplasm. The multiple genes that have arisen from various genome duplication events have evolved different

structure, function, and regulation (Markert, Shaklee, and Whitt,

1975).

1 c 1.3.2 Genetic Interpretations

As allelic variants are expressed codominantly (glossary, appendix

1), identification of genoyptypes and the counting of allelic

frequencies can be made directly from the gel staining pattern and

inferences can be made from this information.

Genetic Variability: Allelic frequency information from isozyme data

can be used as an indicator of genetic variability within

populations. This type of information can be vital In management

schemes that rely to a large extent on hatchery stocking. Mean

heterozygosityH, the average proportion of heterozygotes per locus

(appendix 2), is used as an index of gene diversity. Also used is

proportion of loci polymorphic P. To get a significant figure of

genetic diversity a large number of randomly chosen loci should be

used and even here true genetic variability will be underestimated.

Lewontin (1974) suggests that only one third of amino acid

substitutions will be detected as net charge differences by electrophoresis.

Systematics: Allelic frequencies can be used as an important tool in both intra- and interspecies systematics. With extensive data

indices such as Nei's (1972) genetic identity coefficient (I) or genetic distance (D) can be calculated. Dendrograms can be constructed from these values that can show degrees of genetic divergence and evolutionary relationships. These values are also used as an evolutionary timepiece, where time varies directly with genetic distance (D) (see appendix 2). All these indices are based

17 on the assumption of protein evolution occurring at a stochastically constant rate and not being under the' influence of natural selection.

This assumption is not quite valid (Dobzhansky et al., 1977), however

with enough loci these coefficients may be used to give rough figures

to relative rates and degrees of genetic divergence. Besides these

coefficients, divergence from expected genotypic distributions (see

below) and even consistently significant frequency differences at a

single locus can be regarded as positive evidence of some degree of

genetic differentiation between groups (Utter etal., in press).

Hardy-Weinberg Equilibrium: - This states that in the absence of

selection forces the distribution of genotypes in a randomly mating

population at a polymorphic locus is based on the square of the

allelic frequencies.

If these frequencies differ from the expected H.W.E. distribution

it can be assumed:

Strong selection pressure (pollution, angling, etc.) is

acting disproportionally against certain genotypes, or

The population is not panmictic and some kind of

population segregation is indicated. This assumption may be valid if

there is a disproportionate number of homozygotes. or

Effective breeding in the population is carried out by

only a few individuals, or

Immigration or emigration of particular genotypes is

occurring.

Details of techniques, applications and theoretical considerations

of electrophoresis are given by Harris and Hopkinson, (1976), Smith,

(1968), Ferguson, (1980) and Lewontin, (1974).

0 1.4 ISOZYME RESEARCH IN SALMONIDS

Salmonid fishes have been used in genetic and biomedical research

programmes to study the nature and development of isozyme expression

(Markert eta]., 1975 ; Bailey and Lim, 1975), as well as the genetic models of segregation and linkage which they follow (May, Wright, and

Stoneking, 1979)

This and related research has revealed a large amount of duplicate

gene expression (e.g. five genes for LDH instead of the three of most

teleosts). This biochemical genetic evidence, together with

cytological evidence (chromosome arm number and genome size is

approx. double that of related species), establishes a case for a

polyploid event early in salmonid evolution (Ohno, 1970). The

ancestral tetraploid genotype has, over the course of evolution, lost

nearly 50% of the duplicate gene expression and the evidence is that

those retaining this expression now segregate in normal disomic

fashion (Allendorf, Utter, and May, 1975).

Duplicate loci expression allows for a "fixed heterozygosity" with

associated advantages of hybrid vigour without the consequences of

increased segregational load. This built-in heterosis and

evolutionary potential that polyploidy bestows gives salmonids the

genetic back-up to "considerably enhance their adaptive potential"

(Wilkins, 1972b). The evolutionary potential is also indicated in

the highly variable nature of salmonidkaryotype expression. Closely

related species that produce natural hybrids differ quite

significantly in chromosome number, S. salar and S. trutta by about

twenty, and there is even a good deal of intraspecific variability. Chromosomal rearrangements and divergence has been noted as playing

one of the major roles in speciation (White, 1978). In referring to

the complex mosaic of closely related forms, with diverse

morphologies and ecologies of the white fishes (genus ),

Behnke (1970) states that early systematists felt there must exist

some sort of "essence in their genomes which permits enormous

phenotypic plasticity and/or extremely rapid genotypic evolution".

Wilkins (1972b) claims that the endowment of the tetraploid genome may be this "essence".

1.4.1 Research on Pacific Salmon and Trout

The impetus of the biomedical research programmes on isozyme expression and the necessity to assess and identify fish stocks has established population genetics as an important part of fisheries management (reviews: Utter, Hodgins and Allendorf, 1974; Allendorf and Utter, 1978; Utter et al., in press). In over 10 years of research a general perspective of population structures and genetic variability in the Pacific salmonids is begining to emerge. The perspective outlined in these reviews is given below.

Temporal Stability of Allelic Frequencies:

Isozyme data has been accumulating for just over 10 years and a pattern of temporal stability seems to be emerging. This is to be expected if isozyme data has a valid genetic basis and no strong selection forces are in operation. Variations in temporal stability have been attributed to stocking of hatchery fish from exogenous sources.

2n I Spatial Variation:

Patterns that do emerge of allelic frequency distribution are primarily geographic. This follows the expected model for migratory species that show strong homing instinct to natal spawning streams and as a rule "genetic similarity tends to increase with geographic proximity" (Utter, 1980). This model does not always work however; in rainbow trout, significant differences at two loci (TFN and LDH-4) exist, that seperate the Pacific coast stocks (both rainbow and steelhead) into a coastal and interior form. The boundary appears to be based on the Cascade Crest, which at one time would have divided the Pacific Ocean and an inland sea. These forms now share the same drainage, but evolutionary divergence during the glacial obstruction by the Cascades has been maintained with little or no gene exchange.

Geographic proximity still appears to be the main criterion for genetic divergence. As more loci are studied it is becoming possible to substructure populations into different drainage tributaries and finer geographic subdivisions.

Ecological and Seasonal Variation:

Allelic frequencies of different life history patterns such as

"steelhead" vs. "river resident" or "summer run" vs. "winter run" do not indicate that they are segregating populations. Chilcote,

Crawford and Leider (1980) concluded from data at 6 loci that genetic differences between summer and winter running steelheads are no greater than fluctuations between year classes, and that separate stock status between these "races" (Smith, 1969) needs to be reevaluated. Allelic frequencies of resident and migratory

21 (steelhead) rainbow populations in a region were invariably similar, and both forms showed the same geographic frequency distribution.

The only case where allelic frequency data supports segregation between resident and anadrornous fish, is between a "kokanee" population and anadromous sockeye from Issaquah Creek, Lake

Washington (Utter etal., in press).

Genetic Variability:

Interpretation of H (average heterozygosity) values have to be made with some caution, especially for interspecies comparisons

(Lewontin, 1974; and 1.5), but Allendorf and Utter (1978) present data that they feel has been historically consistent enough for general trends to be observed. From this data (table 1) they observed that rainbow trout, one of the most successful and adaptive of salmonid species, has a significantly higher H than the Pacific salmon (Oncorhyncus) species. A potentially more profitable use of H is with populations within a species. In comparisons of 41 natural and hatchery populations, Allendorf and Utter (1978) showed no great loss of genetic variability except in one population at the

University of Washington hatchery. Possible loss of genetic variability in hatchery fish is discussed more fully in 4.4.'

General points made by Utter etal. (in press) in their review on population structures of seven Pacific coast salmonid species include;

- "the uniqueness of population structuring within species" and that inferences about population structuring in a species cannot be made from genetic data of even closely related

01) Table 1 - Average Heterozygosity

Average Heterozygosity in Nine Species of Salmonids

Number of Species Common name populations H Range of H

Oncorhynchu3 0. gorbuscha Pink salmon 6 0.039 0.032-0.047 0. keta Chum salmon 5 0.045 0.043-0.048 0. kisutch Coho salmon 10 0.015 0.000-0.025 0. nerka Sockeye salmon 10 0.018 0.008-0.024 0. tshcaytscha Chinook salmon 10 0.035 0.024-0.052

Sa imo S. apache 1 0.000 S. ciarki Coastal form 6 0.063 0.022-0.077 Interior form 2 0.023 0.021-0.025 S. gairdneri Rainbow trout 41 0.060 0.020-0.098 S. salar Atlantic salmon 2 0.024 0.020-0.028

from Allendorf and Utter, 1979.

23 species, i.e. generalized conclusions are not reliable,

- protein variation in salmonid fishes seems to be distributed by geographic seperation and random processes. One of the key debates of modern biology has been whether these protein polymorphisms are influenced by natural selection or are adaptively neutral and distributed by random processes and drift. Natural selection is thought to predominate in large randomly mating populations; whereas drift and chance are most likely effective in determining the pattern of distribution in small populations (Nei,

1975). The population geneticists studying the Pacific salmonids have concluded that random events predominate in these species. The evidence on which they base their view includes;

distribution patterns reflecting geographic rather than environmental variables,

the distinctive allelic frequencies of even and odd year classes of pink salmon (Oncorhyncus gorbuscha) that spawn in the same river on alternate years,

the consistency of frequencies among age groups, year classes and generations in closed" populations,

the influence on gene frequencies of transplanted fish on future generations in a population.

Though recognizing the caution given by Utter et al. (in press) that generalized assumptions from isozyme data are not reliable for even closely related species, a preliminary model can be worked out for population structuring in brown trout from the Pacific salmonids data.

1- Populations are based on geographic barriers. This may not just include major river drainages and lakes, but can often be

24 subdivided further to individual tributaries.

Degrees of relatedness of most populations are in most cases based on geographic proximity.

Significant differences in gene frequency distributions between geographically close populations is often an indication of past seperation, usually caused by glacial events.

Different ecological forms, anadromous / non-anadromous, early / late run, etc., often arise from the same population and thus do not appear to be different stocks.

Protein polymorphisms appear to be neutral and are distributed by drift on geographically isolated populations.

Inadequacies to this model quickly appear when it is looked at in detail. Selection may play a more active role than was thought

(3.1.2). Sympatric populations of a small lake show a remarkable divergence (3.1.3). Phenotypic forms show genetic divergence

(3.1.1).

25 1.4.2 Research on Atlantic Species of Salmo

Research on Atlantic species has lagged behind that of the Pacific salmonids. This is in part due to the expense of gaining material, especially of the more commercially important species S. salar, and also because of the different resource ownership rights and access to fish for sampling.

Wilkins (1972) has reviewed work on the Atlantic salmon, and details of genetic variation are gradually accumulating (Payne, 1974;

Khanna et al. 1975; Cross and Ward, in press; etc.). Of particular interest for the segregation of Atlantic salmon populations is the work of Payne, Child and Forrest, 1974; and Child, Burnell and

Wilkins, 1974. These authors concluded that at least two races of salmon could be differentiated in the British Isles based on frequency differences at a transferrin allele. Payne et al. (1971) postulated that this difference arose from isolation and recolonization after glacial recession. Cross, Healy and O'Rourke

(1978) have found significant differences at two enzyme loci between populations of two rivers in the south of Ireland indicating geographic structuring. In comparisons between generations in one of the rivers no marked difference occurred suggesting temporal stability.

Research on Brown Trout

Brown trout has been used in some of the Pennsylvania and New

Zealand work on isozynie expression (e.g. Wright et al., 1975; Lim et al., 1975) but little had been done on its population genetics until

26 a recent series of papers from Sweden (Allendorf et al., 1976;

Allendorf et al., 1977; Ryman et al., 1979). From these papers and work currently being carried ou t in Northern Ireland (Ferguson,

1980), it would appear that some interesting structuring of populations occurs in brown trout (sections 3.1.2 and 3.1.3). An assessment of this work is given in parts of the Results (section 3) and Conclusions (section 4).

27 1.5 LIMITATIONS AND OBJECTIVES

Isozyrne data can be a highly useful tool for integrating

population genetics into fisheries management, but there are some

cautions. Perhaps the first is in relation to what Harris and

Hopkinson (1976) describe as secondary isozymes. This is a class of

isozyme expression that is generated by various "post-translational"

changes and it is difficult to assign a genetic basis to this

expression. Often their presence or absence depends on the buffer

system used or adequate saturation with coenzyme. This makes pH and

temperature control an essential technical requirement of this work.

Because such artifacts can often be present in gel staining patterns,

it is necessary that good genetic models, that have been

substantiated by inheritance work for Mendelian segregation, be

worked out. This is a difficulty with brown trout, as most of the

genetic models of protein variation are based on related species,

especially rainbow trout. Although protein sequences are homologous

in related salmonids, ambiguity can exist about polymorphic proteins

if not validated by inheritance work.

Another difficulty when surveying protein variation is to ensure a

reasonably unbiased and reliable effort. This requires (Lewontin,

1974):

Adequate sample size in the of 50 wild genomes per

1 ocus.

That genomes be tested from natural populations.

A large sample of loci (one of the most important criteria).

A diverse sample of loci coding a variety of enzymatic functions.

5- An unbiased sample of loci that are not chosen because they are polymorphic.

Isozymes are expressed not only in blood and muscle but also

brain, eye, liver and various tissues that require the sacrifice of

the . Obtaining and killing large numbers of fish is

difficult; moreover resource owners, quite understandably, are

reluctant to allow large numbers of fish to be taken for research

purposes.

A final limitation is the nature of the small independant research

effort. Gathering of equipment, learning of techniques, often by

trial and error, is a time consuming process. Interpretations of

complex genetic models from banding patterns can be quite difficult

when relying on published material. The independant researcher is at

a disadvantage in not having the knowledge and technical competance

built up in the larger continuous research effort.

1.5.1 Objectives

No qualitative differences of isozyme expression are likely to exist within a species (there is a notable exception in a lake

population of brown trout in Sweden, see 3.1.3). What may exist are

differences in frequencies in the expression of protein

polymorphisms. If frequency differences are significant then it may

be seen as evidence of population structuring and breeding

segregation. The assessment of population structuring within a

species such as brown trout is then a statistical exercise. So

29 besides data from Scottish samples, evidence from the growing literature is presented in assessing the objectives.

Final objectives were set;

1-(section 3) to survey isozyme expression in samples from the Tweed over a broad range of isozyme loci and compare four sites (2.1) for allelic frequencies at 3 polymorphic loci (LDH-5,

AGP-2 and CPK-1),

2-(section 4) to investigate the evidence from protein variation of population structuring and the relationships of the polytypic forms of brown trout. MATERIALS AND METHODS.

31 Figure 1 - Materials

a') Comparison of wild fry (above) with one of similar age kept in hatchery facilities of the Department of Forestry and Natural Resources showing environmental induced colour variation.

b) Comparison of young trout with salmon parr. 0.

-

1 Figure 1 Materials

c) Comparison of sea trout with Atlantic salmon (grilse)

d) Comparison of male sea trout with male brown trout too 440

•'ii : ;: . ' f•t• " j -1 t, " " 4 ezc AF : I • ne

I m

: Figure 1 Materials

e) Fyke net on Meldon Water (tributary of the River Tweed) catching migrating smolt.

f) Seine netting on Loch Ullambrasco, Scaliscro, Lewis -

• -V V.. . - - --V.. - VV • VV.. V ___

AO

JF . 4W

V Figure 1 Materials

g) Electrofishing for parr and smolt on upper tributary of the Tweed. I Not

4 I Figure 1 Materials sc

h) Sampling sites in Scotland 2.1 MATERIALS

Samples were obtained from four sites in Scotland using a variety of methods of capture; ANG-angling, ELC-electrofishing, FYK-fyke net, and SEI-seine net.

TW River Tweed

- Samples were caught by FYK and ELE in some of the up-river burns; Glen Sax, Meldon Water and Kirk Burn and by SEI down-river to catch migrating smolt. Most samples were smolt but also included were parr.

NE North Esk, nr. Montrose, Angus

- Samples are all down-river migrating smolt caught in the Dept. of Ag. and Fish. trap at

Montrose. These fish originate from unknown, and most likely several spawning tributaries.

BR Braid Burn, Edinburgh.

- Samples are all stream dwelling brown trout, there is no access to anadromous fish. All fish were caught by ELE

SC Loch Ullambrasco, Scaliscro, Lewis, Outer

Hebrides.

- Samples are lacustrine brown trout.

No access to anadromous fish. Samples were caught by ANG and SEI. 2.2 METHODS

Methods employed were similar to those described by Smith (1976) and May et al. (1979) with some alterations recommended by A.

Archibald of the A.B.R.O. Institute, Edinburgh. Quarter inch (6 mm.) glass plate and glass strips were used to form the mould which was held together with necol adhesive cement. Connaught hydrolysed starch was used throughout (Connaught Laboratories Ltd., Toronto,

Canada) at 13 w/v (39.0 gms. in 300 ml. of the appropriate buffer).

The mixture was heated to boiling with constant stirring and degassed with a water aspiratorvacuum for approx. 40 seconds. The gel was then poured into the mould and allowed to cool.

Tissue; muscle, liver, heart, etc. were placed with a roughly two times equivalent of 0.05 M Tris-HC1 buffer pH 7.6 in plastic centrifuge tubes and crushed with a glass stirring rod. The homogenate was spun down briefly in a centrifuge. Wicks (3 mm. by 8 mm.) cut from "Whatman's No. 17 Chromatography Paper" were dipped into the supernatant and placed along an edge cut about 5 cm. from one end of the gel. Electrophoresis was carried out in a Shandon

"Universal" tray using sponge washing up cloths as current bridges.

Migration was observed with Bromophenol blue indicator on the

right-hand most wick. The buffers and staining system were those

used by Allendorfetal., (1977). Gels were sliced on a Shandon gel

slicer and placed in plastic freezer trays for staining. Tops and

bottoms were discarded and three slices could be taken from the gel.

After staining the gels were fixed with a 1:4:5 solution of acetic

acid, methanol and distilled water. Banding patterns were then noted

and recorded. Figure 2 Methods

a) Starch boiling under vacuum

b) Pouring hot starch into mould 11t ,o',

4mvp "Oft Figure 2 Methods

c) Placing sample wicks on gel

d) Removing sample wicks after ten minutes of running AV

ArT-

- Figure 2 Methods

e) Cooling with ice pack

f) Slicing gel - OF

- -

-

• - L

Will 2.2.1 Buffer Systems

Electrophoresis Buffers:

Ridgway etal. (1972).

Electrode: 0.06 M lithium hydroxide - 003 M

boric acid, pH 8.1.

Gel: 0.03 M Tris - 0.005 M citric acid, pH

8.5

Gels made using 99% gel buffer - 1% electrode buffer.

Clayton and Tretiak, 1972

Electrode: 0.04 M citric acid, pH 6.1 adjusted with N-(3-Aminopropyl)-morpholine.

Gel: 1:20 solution of electrode buffer, pH

6.0.

Staining Buffers:

0.2 M Tris-I-IC1, pH 8.0.

A solution of 1:4 stain buffer I and water.

The A buffer system gel mixture, i.e., 99% A

gel buffer and 1% A electrode buffer.

41 2.2.2 Staining Systems

Staining relies on the visualization of the site of specific enzyme activity and requires 4 components besides the actual ENZYME present in the gel matrix. An example with lactate dehydrogenase LDH is as follows:-

- LDH acts on its SUBSTRATE, lactate with a COFACTOR, to produce pyruvate, NADH and an extra transient hydrogen ion.

- This hydrogen ion reduces phenazine methosulphate

(PMS) which is an intermediary hydrogen ion carrier to the STAIN a yellow soluble tetrazolium salt, nitro-blue tetrazolium (NBT.) or methyl thiazolyl tetrazolium (MTT); which when reduced by the hydrogen ion turns to a dark blue insoluble formazan dye.

- This hydrogen ion transfer is buffered in a STAIN

BUFFER

Details of stain components are given in table 2.

I,

Table 2 - Staining

Enzyme Stain Cofactor Stain buffer Substrate

Alcohol dehydrogenase ADH NBT/PMS MAD III lOmi 95% ethanol

cz-glycerophosphate dehydrogenase AGP NBTJPMS MAD III 1 g DL-a-giycerophosphate

Creatine phosphokinase CPK VISUALIZED WITH GENERAL PROTEIN STAIN

Isocitrate dehydrogenase IDH NBT/PMS NADP II 200mg DL-isocitric acid

Lactate dehydrogenase LDH NBT/PMS NAD III 25nil 0.5MDL-Na-lactjc acid

Malate dehydrogenase MDH NBT/PMS NAD III 25m1 0.5MDL-Na-rnalate pH 7.0

Malic enzyme ME NBT/PMS NADP III 25m1 0.5MDL-Na-malate pH 7.0

6-phosphogl uconate dehy droge nas e 6PGDH NBT/PMS NADP II 100mg Ba-6-phosphogluconic acid

Phosphoglucose isomerase PGI NBT/PMS NADP III 25mg Na-fructose-6- phosphate 10 G6PDH

Phosphogiucomutase PGM NBT/PMS NADP II 300mg K-glucose- i -phosphate 50 G6PDH 5 drops a-D-giucose-1,6 di phosphate

Sorbi tol dehydrocienase SDH NBTJPMS MAD I lOOmg'D-sorbitol +2.5mc! MTT

Superoxide dismutase SOD VISUALIZED AS WHITE "NEGATIVE" BAND WHEN STAINING FOR OTHER ENZYMES, e.g. ADH, AGP or MDH.

43 2.2.3 Nomenclature System

The profusion of nomenclature systems in the literature, see

(Wright et al., 1975), has added to the confusion of the outside reader. Fortunately the system proposed by Allendorf and Utter

(1978) and extended by May, Wright, and Stoneking (1979) appears to be gaining general acceptance and is reasonably easy to follow.

Enzymes are abbreviated usually to three letters, as LDH for lactate dehydrogenase. Underlined (or italicized) it refers to a gene locus coding for that enzyme. Where multiple loci exist that code for an enzyme a hyphenated numeral follows the abbreviation, the numbering from most cathodal to anodal. LDH-5 is the mostanodal of the five gene loci that express LDH. Alleles are specified by their relative mobility on the gel in comparison to the most common type which is designated "100". The most common allele at the fifth LDH locus is

LDH-5(100). A slow variant that migrates only half the distance would be referred to as the LDH-5(50) allele and a heterozygous individual containing both alleles would be designated LDH-5(50/100).

Where a duplicate loci pair exist that migrate at the same rate and a polymorphism is expressed from one or other of this pair, the variant allele is designated as MDH-3,4(125). In some cases, particularly

LDH and MDH, I have extended this nomenclature system to include some of the alphabetic designations used in the earlier literature (Wright et al., 197.5). These are usefull to clarify the differences between certain types of enzymes. LDH-A is the type of LDH found in anaerobic white muscle tissue and is coded by the two loci; LDH-1 and

LDH-2, forming two polypeptides LDH-1 or Al and LDH-2 or A2.

Problems can arise using this nomenclature system when researchers

/1,1 use different buffer systems that give different migration rates.

However it does provide a unifying and simple system on which to build and standard references are becoming established (May et al.

1979).

45 It nriit -1- c-

Twelve isozyme systems were attempted that are coded from over twenty loci. The Tweed samples (TW) were run and stained for all systems. The other sampling sites were stained to compare allelic frequencies at LDH-5, AGP-2, and CPK-1; though freezer failures jeopordized some of the samples. The isozyme systems are described as to; their biochemical basis and function; the genetic models that are assumed and polymorphisms that have been reported; the occurrence of any polymorphisms in the Scottish samples and allelic frequency information if possible; and any information on the nature of isozyme variation, population structuring, etc., that may be given by this system.

46 3.1 LACTATE DEHYDROGENASE

Lactate dehydrogenase (LDH)(L-lactate: NAD oxidoreductase, E.C.

1.1.1.27) is a tetrameric enzyme present in all vertebrate cells. It

provides an important intermediate reaction in cell metabolism

between anaerobic glycolysis and aerobic respiration. The

interconvertible reaction of pyruvate to lactate which LDFI catalyses

provides, simultaneously with the accumulation of lactate, the

oxidation of NADH to •NAD which powers anaerobic glycolysis thus

providing energy under oxygen stress conditions. The reverse

reaction must also operate efficiently to oxidize the harmful excess

lactate and remove it through aerobic respiration or through

gluceonogenesis (Lehninger, 1971).

glycolysis

NAD NADH

lactate pyruvate

LDH

aerobic respiration

The dual nature of this reaction has evolved in LDH a number of molecular forms or isozymes that are highly tissue specific. By

varying their affinity for the substrate and resistance to product

accumulation these molecular forms have adapted to the catalytic

47 needs of the various tissues.. There is also a good deal of variation of these isozymes between and within species, more than one might expect from an enzyme that is so fundamental to cell metabolism

(Markert and Ursprung, 1971). This has made LDH one of the most widely studied and interesting isozyme systems and much of this interest has centered on salmonid fishes.

The general vertebrate model for LDH is of two autosomal gene loci that determine two polypeptides, A, and B; these form five tetrameric

isozymes; AMA, AAAB, AABB, ABBB and BBBB (Markert and Ursprung,

1971). Most teleosts also produce a third highly anodal polypeptide that is specific to eye tissue. It is hypothesized that this gene

LDH-E arose from the LDH-B gene by tandem duplication quite early in

teleost evolution (Whitt, Miller and Shaklee, 1973).

Salmonid fishes have an exceptionally large number of

electrophoretically distinguishable isozymes. This has presented

difficulty in describing a model for LDH genetics, with suggestions

ranging from four to ten structural loci (Bailey et a]., 1976). The

consensus model now is that there are five structural gene loci

(Wright et al., 1975; Bailey et al., 1976; Lim and Bailey, 1977)

(figure 3a). All five common alleles have unique electrophoretic

mobilities and for the most part have evolved tissue specificity.

The five loci can be traced back to the polyploid event at the early

stages of salmonid evolution on the A, B, and E teleost loci. The A

and B loci have retained duplicate gene expression while the E locus

has lost it. LDH - Genotypes

A - shows expression of fast allele at LDH-5 - LDH-5(105)

B - common hornozygotes LDH-5, LDH-4, LDH-3 shown in eye tissue

D - full expression of LDII-4, LDH-3, LDH-2 and LDH-1 in white muscle tissue

E - muscle extract showing no LDH-1 expression Figure 3a - LDH Genotypes

26 LDH

-I . .

LOH-5 LDH-L LDH -3 LDH-2 1 LDH-1 0 A B B 0 E

49 LDH - Genotypes

3b - 1 - the fast eye phenotype is shown (A)

this variant allele is designated LDH-5(105)

- because migration differences are only slight it is difficult to differentiate genotypes

- allelic frequencies are calculated using the

square root of the frequency of the common

phenotype. (This assumes H.W.E. distribution)

3b - 2 - Atlantic salmon eye phenotypes (F) are

compared with the variant trout phenotype

3b - 3 - Common and variant eye phenotypes B and A and

the homotetramer of LDH-4 found in liver (G) Figure 3b - LDH Genotypes LDH-5 (eye) variants

1 A \ LDH

2

ft 44M -Ift Ow -

InIt el ow w ow I-

ABBAF BB F

3

LOH

,low

50 LDH - Genotypes

3c - 1 and 2 - this shows the variability of

expression of LDH-1

- if this is a genetic polymorphism

D would represent the common homozygote, E the

variant homozygote, and presumably H represents

the heterozygote.

- in both these figures the

samples (E) of the variant homozygote were

fry of less than 8cm. Figure 3c - LDH Genotypes

LDH-1 (white muscle) variants

1

24 1 2 LDHJ 'If lose D D H E D H DOHD

2 2g mo 3

0 HE H Ei

51 N Table 3

LDH-5 Allelic Frequency

fast phenotypes frequency Sample N S.E observed iuioout allele

TW 75 4 0.973 ±0.013

NE 35 1 0.986 ±0.014

BR 30 0 1.00

Sc - freezer failure invalidated samples -

fast phenotypes observed are assumed to be heterozygotes. LDH-5 (105/100)

52 3.1.1 LDH-5

LDH-5 is the gene locus that codes for the LDH-E polypeptide in salmonids. This highly negatively charged isozyme is synthesized in regions of the nervous system concerned with vision, particularly the neural retina (Whitt, Miller and Shaklee, 1973). LDH-E has a very high affinity for its substrate and may play a part in providing NAD for retinol dehydrogenase in the visual cycle (Whitt, Miller, and

Shaklee, 1973; Wald, 1968) under low pyruvate concentrations; or because it appears to have high inhibition to lactate, and this tissue is very aerobic, the reaction is primarily in the other direction, scavenging exogenous lactate for energy use (Whitt, Miller and Shaklee, 1973).

Genetic variants at this locus have been reported in

(Wright and Atherton, 1970), rainbow trout (Wright et al., 1975) and

Atlantic salmon (Khanna et al., 1975). A faster allele has been reported in Scandinavian brown trout (Allendorf et al., 1977) designated LDH-5(105).

This polymorphism was detected in Scottish fish but because migration differences are only slight and resolution is poor, positive identification of genotypes is difficult (figure 3b).

Allelic frequencies at this locus are based on the square root of the frequency of the slower phenotype, the common homozygote, assuming a

H.W.E. distribution. The faster phenotype is marked as being either the fast homozygote genotype or the heterozygote. In low frequencies it is most likely the heterozygote.

53 This polymorphism occurs rarely in the three east coast sites; N,

NE and BR, are all less than 3% (table 3). No figure could be obtained for the Isle of Lewis site (SC) due to prolonged storage and freezer failure. In Lough Melvin, western Ireland, Ferguson (1980) showed a significant variation from Hardy-Weinberg distribution at this locus and another locus (PGI-2) (he was able to differentiate heterozygotes from fast (105) homozygotes). When he split the 459 trout into phenotypes- ferox, gillaroo, and sonaghen he found genotype frequencies fit into a H.W.E. distribution. In particular had a high allelic frequency for the 105 allelle, aprox.

60%. Ferguson concluded that these trout phenotypes showed evolutionary divergence (see section 4.3). This is one of the recently published works on brown trout that shows segregation of sympatric populations.

Ferguson (pers. comm.) has found this polymorphism to occur in high frequencies in trout of the west coast of Ireland. This polymorphism occurs rarely in most Swedish populations with the exception of Lake Lulejaure, a large lake in north central Sweden

(Ryman and Stahl, in press)

The fast phenotype of brown trout migrates at the same rate as the common phenotype of Atlantic salmon. Published information by Khanna et al. (1975) has shown Atlantic salmon to have a variant eye LDH

'which corresponds very closely to the common trout type (figure 3b).

54 3.1.2 LDH-B

LDH-3 and LDH-4 are the gene loci that code for the LDH-B polypeptide in salmonids. LDH-B is usually expressed in tissue that depends on aerobic metabolism, such as heart tissue, as opposed to

LDH-A which .is associated with anaerobic conditions in such tissue as white muscle. Tissues of most vertebrates balance their catalytic

requirements by favouring various A-B heterotetramer combinations.

In salmonid fishes this coverage seems, to have been replaced by a

B1-B2 series expressed from the LDH-3 and LDH-4 loci (Lim et al.,

1975). The B2 polypeptide of LDH-4 is halfway in catalytic

properties between LDH-3 (Bi) and LDH-2 (A2) and has taken on the

role of the A polypeptide in regulating the catalytic reaction in

tissues that might experience moderate anaerobic conditions (Lim et

al., 1975). It has however not been able to replace the A

polypeptide in tissue where reaction favours extreme anaerobic

conditions and excess lactate accumulation such as in white muscle

tissue.

No variants of these loci have been reported in brown trout though

the variation at LDH-4 (the homotetramer expressed in liver tissue)

in rainbow trout has some relevance to the nature of isozyme

variation. The high frequency of a slow allele in the upper parts of

the Columbia and Fraser river drainages east of the cascade crest,

led Allendorf and Utter (1978) and Utter et al., (in press) to

conclude that there was evolutionary divergence between an Inland and

Coastal form of rainbow trout based on geographical seperation

(1.4.1). The view that these authors express (Utter et al., in

press) is that in salmonids genotypic distributions of natural

55 populations would be principally a reflection of random factors".

The higher incidence of the slow variant in interior streams has led some researchers (Northcote et al., 1970; Huzyk and Tsuyuki, 1974) to speculate that there might be some adaptive feature of this allele to suit fast flow and higher temperature conditions of headwater streams. This polymorphism thus, can be looked at in the context of the debate between selection vs. neutrality in polymorphic protein loci. -

Tsuyuki and Williscroft (1973) found a 3.6 times greater conversion rate of lactate at pH optima for the slow B2 homotetramer

and predicted this might confer superior swimming endurance. In later tests Loon lake rainbow trout fry did indeed show that the slow homozygote had 2.3 times greater swimming stamina than the "100"

homozygote (Tsuyuki and Williscroft, 1977); from this study they concluded that stocking programmes for resident rainbow trout should take into account the superior swimming stamina of the variant

homozygote. Later tests when the fish were 27-29 months, indicated a

reversal in swimming endurance with the slow homozygote showing

erratic behaviour; and the same tests on coastal and interior

steelheads showed no significant differences in phenotypes.

Klar, Stalnaker and Farley (1979) conducted similar tests on mature rainbow trout under saturated and limited oxygen

concentrations. Under saturated conditions no significant

differences in swimming performances was noted for the different

phenotypes. In the limited oxygen test though, the slow hornozygote

performed the worst. In another study these authors (Kiaretal.,

1979b) demonstrated that in oxygen stress conditions the slow

56 homozygote fish exhibited convulsive behaviour and suffered

significant mortality. These authors, citing an earlier work (Kao

and Farley, 1978), observed that the slow B2 isozyme showed

characteristics of A-type LDH and felt that under low oxygen

concentrations it would shift into lactate production. As this

isozyme is expressed strongly in the brain, excess lactate production

to toxic levels could explain this convulsive behaviour; this erratic

behaviour was also noted for the older slow homozygotes in Tsuyuki's

and Williscroft's (1977) study. Juvenile fish might supply adequate

oxygen as larval hemoglobin has higher oxygen affinity than adult

hemoglobin (Kiar et al, 1979a) and young slow homozygotes might

indeed be superior in their swimming stamina as proposed by Tsuyuki

and Williscroft, however it appears that this phenotype is at a

distinct disadvantage as it approaches maturity.

These studies and. others (Redding and Schrek, 1979) of this

polymorphism give strong evidence for selective features in protein

polymorphisms. If natural selection maintains the high level of this

allele in the "interior" rainbow trout populations it is not likely

to be because of advantage for the homozygote but through some more

complex balancing selection.

57 3. 1.3 LDH-A

LDH-1 and LDH-2 are the duplicate loci pair that code for the LDH of white muscle tissue, LDH-A. Structural divergence between the two polypeptides, Al and A2, is in the order of 12 to 25 residues per subunit in quinnat salmon, this is comparable to the divergence between the Bi and B2 polypeptides (Lim and Bailey, 1977). But unlike the B subunits the A have not evolved into different roles and appear to be catalytically equivalent.

A number of authors have indicated a poor and slow expression at the LDH-1 locus. Wilkins (1971) states that small Atlantic salmon

(<8cm.) were poor in expressing muscle tetramers. Utter, Allendorf, and Hodgins (1973) report poor expression of the Al homotetramer and

Jones (1976) found no expression of the Al homotetramer until 123 days post fertilization in Atlantic salmon.

After quantitating techniques were applied to quinnat salmon muscle isozymes, it was found that the A2 and Al polypeptides occurred in a 4:1 ratio (Lim and Bailey, 1977). They hypothesized that mutations are accummulating in the regulatory DNA preceding the structural gene that expresses the Al polypeptide. Although there has been resistance to loss of expression at this duplicated locus

(if the Al polypeptide offered no advantage at all it should have drifted into disuse a long time ago), there has apparently been no selective brake to mutations that affect its levels of synthesis.

A polymorphism at LDH-1 was noted in Swedish brown trout by Allendorf et al., (1976). As no expression was observed in this polymorphism it was concluded that the variant allele was a null allele or migrated at the same rate as LDH-2. They chose the latter for descriptve purposes, designating it LDH-1(240).

This polymorphism was noted in the Scottish fish samples and

LDH-1 expression seemed quite variable (figures 3a & 3c). Given the apparent difficulty of expression of LDH-1 and the conclusions of

Lim and Bailey (1977) this variant could be a null allele; due either to increasing regulatory mutations or a mutation in the structural gene that makes it non viable. Poor expression in the

Scottish samples appeared to have an ontogenetic factor, null homozygotes always appeared to be fish of less than 8 cm., although heterozygotes did appear in smolt. This could be due to my sampling as most fish were small and immature, and Gunnar Stahl (pers. comm.) found variant null homozygotes in 5 year old fish in Sweden.

An interesting population of this variant allele has been reported in a Swedish lake, in which half the fish sampled are the variant homozygote and the others are the common homozygote. In over 300 samples from this lake system no heterozygotes were found

(Allendorf et al., 1976; Ryman, Allendorf and Stahl, 1979). Using the two homozygotes as markers, these researchers were able to

seperate the Lake Bunnersjoarna trout into two reproductively

isolated demes which have statistically significant allelic

frequency differences at five loci (table 4) as well as differences

in size distributions. Table 4 - Allelic Frequencies Lake Bunnersjoarna, Sweden

Allelic frequencies in demes

Deme I Deme II Locus (allele) LDH-1(1001100) LDH_1(2401240)t

AGP-2(100) 0.976 1.000 4.82*

CPK-1 (100) 0.562 0.293 26.04***

EST-2(100) 0.967 0.886 3.37

LDH-5 (100) 0.872 1.000 12.63***

MDH-4 (100) 0.571 1.000 51.08***

SDH-1 (100) 0.891 1.000 10.58***

SOD(100) 0.989 1.000 0.01

x2 = chi-square values for differences between denies (2x2 contingency tests). Significance; *, P0.05; P0.01; P0.001.

from Rynian, Allendorf and Stahl, 1979.

t This allele is considered now to be most likely a null allele rather than the (240) that was originally designated.

ME Lake Bunnersjoarna is a small oligotrophic twin lake system of about 40 ha. each, connected by a 50 m. long channel. Water is fed into this lake system from a mountain stream and the outlet leads into a large river drainage. The inlet lake is very shallow (0.5 to

1.0 m); the outlet lake is somewhat deeper. This area has very severe winter weather and the lakes are frozenfrom October-June.

The Swedish workers have speculated that the genetic segregation shown might have originated from an early glacial refuge, (deme II - the rare homozygote) and the common trout type, (deme I - the common

LDH) which have retained their isolation since coming into contact after post glacial reinvasion. They have put forward a case for incipient speciation with little genetic divergence (Ryman et al.,

1979).

The difficulty with this view is to find a mechanism by which isolation can be maintained for the period of over 10,000 years since the last glacial recession. Their evidence points to some differential homing to inlet and outlet spawning streams (deme II choosing the inlet stream), but although Stuart (1957) found strong homing in brown trout, it seems unlikely that complete isolation could be maintained for so long. Whether some strong ecological pressure, place of spawning (lake bottom), timing of spawning, etc., comes into play, has yet to be determined.

Perhaps instead of looking at two long seperated demes, we might be looking at a seperation that is more recent. Recolonization of the inlet lake and stream after a climatic catastrophy (with shallow

61 water and long ice cover, winter kills could easily occur) by a few individuals could create a "founder effect". This population

"bottle neck" will cause an initial loss of heterozygosity (White,

1978) as indicated by the allelic frequency data (table 4), where five out of seven polymorphic loci are now expressed as homozygotes.

Although there are difficultieswith this alternative hypothesis, it does avoid the problem of explaining how taking a major step towards speciation, (reproductive isolation), has been maintained in these sympatric populations over such a long period and with very little other genetic divergence.

The genetic nature of this polymorphism needs to be clarified.

Inheritance studies are now being conducted in Sweden and initial reports are that there may indeed be a null allele (Gunnar Stahl, pers. comm.). It may be that polymorphisms for this isozyme reflect regulatory mutations that somehow affect its level of synthesis and perhaps several such polymorphisms exist; some of this LDH-1 isozyme expression may be influenced by ontogenetic development. It is also interesting to note that Mark ert, Shaklee, and Whitt (1975) have presented a model of gene evolution after duplication in which one of the protein products acquires regulatory properties and may eventually evolve into an enzyme of regulatory function.

62 3.2. 1 Al phaglycerophosphate Dehydrogenase

Aiphaglycerophosphate dehydrogenase (AGP; E.C.1.1.1.8) is a dimeric enzyme which catalyses the reaction:

dihydroxyacetone phosphate + NADH aiphaglycerophosphate + NAD

The genetic basis of this enzyme has been difficult to determine due to discrepencies that are buffer related. It has .been recorded

as being coded by three (Engel et al., 1971; Allendorf et al., 1977),

two (May, Wright and Stoneking, 1979) and one loci (Utter etal.,

1973a) in brown trout and related salmonids. The model that can be

interpreted from the banding patterns (figure 4) of a duplicate loci

pair, the anodal one fixed should be treated warily (May, Stoneking, and Wright, 1979). The anodal bands would appear to be buffer

sensitive artifacts. As the genetic structure of AGP is unclear, I have used the designation of Allendorf et al. (1977) in referring to the allelic polymorphism as AGP-2(50). One indication of a valid genetic basis to this polymorphism is that in all four sampling sites banding genotypes segregated quite closely to Hardy-Weinberg expectations (table 5); although this in itself cannot be taken as confirmation of a genetic model (Fairbairn and Roff, 1980; 4.2).

It is of interest to note that the Tweed (TW) and North Esk (NE)

samples, both smolt, have identical allelic frequencies and in both

populations frequency distributions fit very well into Hardy-Weinberg expectations (table 5). This would indicate a fairly homogeneous distribution of this allele throughout the upper spawning tributaries.

63 I

AGP-2 Genotypes

A - AGP-2(1001100) homozygote

B - AGP-2(100150) heterozygote

AGP-2(50150) homozygote

D - expression from swim up fry

dotted lines indicate secondary isozyme expression Figure 4a - AGP-2 Genotypes

A A A B D A

+

WNW 100 mom 50 0- A A B C

64 Table 5

AGP - Genotypic Distribution

Genotype Sample N Allelic frequency 100/100 100/50 50/50 "100" allele

TW 106 92 14 0 0.934 (92) (14) (0)

NE 92 80 12 0 0.935 (80) (12) (0)

BR 82 62 20 0 0.878 (63) (18) (1)

SC 80 48 24 8 0.750 (45) (30) (5)

Expected values (Hardy-Weinberg equilibrium) in parenthesis.

Significances of deviations from H.W.E

BR - x2 = 2.2 p > 0.1

Sc - = 3.0 0.1 > P > 0.05 x2 - test - log likelihood (G) test Sokal and Rohlf (1969).

65 Figure 4b - AGP-2 Allelic Frequencies and Confidence Limits

sites

TW

NE

IMI

Sc

0.700 0.00 0.900 1.00

frequency of lOO" allele

- confidence 1imits for peróentage (Rohif and Sokal, 1969) 3.2.2 Creatine Phosphokinase

Creatine phosphokinase (CPK; E.C.2.7.3.2) is a dimeric enzyme that catalyses the reaction:

phosphocreatine + ADP creatine + ATP

The high energy compound phosphocreatine acts as a reservoir in muscle tissue for phosphate groups that can be transferred by CPK for muscle contraction energetics (Lehninger, 1975). Although it occurs in other tissues it is present in high concentrations in muscle and is readily visualized with a general protein stain such as 0.1% naphthalene black.

CPK provides an example of the difficulty and complexity that can arise in describing genetic models from isozyme patterns. Although. a dimer the banding patterns produced are not consistent with the usual homo-heterodimeric associations. Phenotypes of two to five bands occur in the genus Salmo and both two and three banded phenotypes appear in brown trout (Utter, Allendorf and May, 1979).

Eppenberger et al. (1971) described some epigenetic factor in the determination of CPK banding phenotypes. Ferris and Whitt (1978) found that while heterodimers can form in vitro they do not 'form in vivo. Using both these pieces of evidence and inheritance studies

Utter, Allendorf and May (1979) have put together the following model of CPK isozyme expression in salmonid fishes:

- No heterodimers form between allelic products at a single

67 locus, or between loci.

- Each allele through some stable posttranslational modification

is represented by two electrophoretic bands.

- Muscle CPK is coded by two. loci.

Brown trout electrophoretic phenotypes can be described using this model assuming a fixed anodal CPiK2 and variation at the CPK-1

locus, in which the rare-fast allele products, (each allele having two electrophoretic products) have the same mobilities as the CPK-2

locus products.

Allendorf et al. (1976) designated the fast allele CPK-1(115).

The common homozygote and the heterozygote are both three banded and although differences in intensities can be noted on fresh samples

(figure 5), reliable estimates of allelic frequencies can only be made on the identification of the fast homozygote (the two.banded phenotype). This means if frequencies are low a large sample size must be taken to ensure reasonable counting of the variant allele

(e.g. at p=0.1 variant homozygotes will occur at 1:100). Figures that indicate no CPK-1 polymorphism (table 6) must be treated warily. Frequencies of this polymorphism from the four sites would appear low, though its appearence is quite high in some Scandinavian populations.

The variant two banded phenotype is the same as the common phenotype of Atlantic salmon and Khanna et al. (1976) has noted a variant phenotype in Atlantic salmon that is the same as the three banded phenotype of trout. This parallels the situation in the CPK Genotypes

showing variation at CPK-1

A - Common homozygote CPK-1(1001100)

B - Heterozygote ci-i (100/11.5)

C - Fast homozygote aPK-1(1m/115)

frequency data can only reliably be scored on identification of the fast homozygote (C) hetérozygotë àë notideabte on fresh samples Figure 5 - CPK Genotypes

A C B A

CPK

II --

BC A CPK

+

111 C

-115 ALLELE

- 100 ALLELE 69 Table 6

CPK-1 Allelic Frequency

Sample N "115" homozygotes Frequency observed "100" allele

TW 60 2 0.82

NE 32 0 1.00

BR 30 0 1.00

Sc 36 0 1.00

Frequencies are calculated from the square root of the "115" homozygote.

70 LDH-5 polymorphism. Gardiner (1974) using a general protein stain, set out a method of identifying young fry of salmon and trout based on the two and three banded phenotypes. Although this is a very good method to apply where the CPK-1 polymorphisms are rare, it would not be applicable in populations where the variant alleles occur in high frequencies and illustrates the dangers of using electrophoretic banding patterns without adequate genetic models.

71 3.3.1 Malate Dehydrogenase

Malate dehydrogenase (MDH; E.C.1.1.1.37) is a dimeric enzyme that catalyses the reaction:

mal ate + NAD oxal oacetate + NADH + H

It occurs in two forms in salmonid fishes as with other higher animals; one in the mitochondria and the other in the extra-mitochondrial cytoplasm (supernatant). Three forms of supernatant MDH have been noted (Bailey et al., 1970; Clayton et al., 1975):

MDH-A the liver/neural form coded by two loci designated MDH-1 and MDH-2 by Mayetal. (1979), although in rainbow trout there may be only one locus coding for this form

(Allendorf, Utter and May, 1975).

MDH-B the muscle form also coded by two loci (MDH-3 and MDH-4) whose products migrate equally to form the most anodal electrophoretic band.

And a heterodimeric A-B supernatant MDH.

A slow moving mitochondrial MDH can appear especially after freezing and thawing of extracts and in brown trout appears to migrate cathodally.

Allendorf et al. (1977) reported polymorphisms in the supernatant muscle form of MDH, noted as MDH-3,4 (125)' a fast allele and

MDH-3,4(80) a slow allele. Both polymorphisms appear in Scottish

72 MDH Genotypes

MDH-A Genotypes

A - Common homozygote

B - MDH-2 (100/152) heterozygote

C - MDH-2 (152/152) homozygote

MDH-B Genotypes

D - Common homozygote

F - MDH-3, 4 (125)

G - MDH-3,4(80) Figure 6 - MDH Genotypes

MDH

I11 F -i iJ I L I

~ MDH-34

MDH-2

o - ___ - MDH-1 A B C F C

73 Table 7

iDH-2 Allelic frequency

Sample N Frequency "100" allele S•E

TW 36 Ow ± 0.06

common fast homozygote he te ro zygote homozyoote 100/100 152/100 152/152

17 15 (16.7) (15.7) 36 (3.7) 36 36

(expected values in parenthesis)

As with the AGP-2 distribution (table 5), observed distribution of genotypes fit very well with expected figures of a population in Hardy-Weinberg equilibrium.

74 trout samples (figure 6) though they were only clearly resolved in the amine-citrate buffer system. As the polymorphic genotypes cannot be reliably determined allelic frequencies are calculated from the square root of the 100/100 genotype.

MDH-1 and MDH-2 expressed in the liver and eye are both reported as being polymorphic in brown trout (May etal., 1979), with a fast allele at MDH-2, designated MDH-2(152) and a null allele at MDFI-1.

The MDH-2(152) allele appears in Scottish -fish (table 7) and the null allele may appear but it is difficult to verify as it appears close to the origin and no homozygotes, which would mark out this polymorphism, were observed.

3.3.2 Malic Enzyme

Malic enzyme (ME; E.C.1.1.1.40) is a tetrameric enzyme that decarboxylates malate to pyruvate requiring NADP as a cofactor. It occurs in supernatant and mitochondrial forms (Cross et al., 1979).

Supernatant ME appears as a single anodal band that is most expressive in liver tissue. Mitochondrial ME; ME-1 and ME-2 were noticed as faint bands in fresher samples of heart tissue.

Some sort of polymorphism exists at ME-3, (figure 7) but as only a fast and slow band appears without any obvious heterozygote expression, a genetic basis to this polymorphism must await more rigourous confirmation. A far higher proportion of the slow banded phenotypes appeared in the BR samples, but without a valid genetic model, not a great deal may be interpreted from this.

75 Malic Enzyme Genotypes

A Heart tissue showing mitochondrial ME-1 and ME-2

B ME-3

C Fast variant at ME-3

ME-1 and ME-2 appear as faint bands and often disappear with prolonged storage of gel. They also appear best in fresh samples. Figure 7 - Malic Enzyme Genotypes

ME '

•:7r:!r . T

B B C B C

ME-3

- ME-2

- ME-i

A B C

76 3.3.3 Phosphoglucoisomerase

Phosphoglucoisomerase (PGI; E.C.5.3.1.9.) is a dimeric enzyme that catalyses the isomeration of glucose 6-phosphate to fructose

6-phosphate; one of the initial steps in glycolysis. The model presented by Allendorf et al. (1977) of three loci showing a six-banded fixed heterozygotic effect similar to MDH worked well in gels stained for PGI. A variant allele, either a slow allele at

PGI-3 or a fast allele at PGI-2 was noted. Ferguson notes a fast allele at the PGI-2 locus in Irish fish, designated PGI-2(140), and this is most likely the variant that appears in Tweed trout.

This initial survey would indicate infrequent expression of this polymorphism. Only one sample in thirty showed the variant allele; indicating frequency figures that show it as a rare polymorphism

(less than 2%). Allelic frequencies in the Lough Melvin trout

(Ferguson, 1980) are in excess of 30% and Ferguson (pers. comm.) has found very high frequencies in populations in southern Britain.

-7-7 3.3.4 Phosphoglucomutase

Phosphoglucomutase (PGM; E.C.2.7.5.1.) is a monomeric enzyme that

catalyses the reaction:

glucose 1-phosphate , ' glucose 6-phosphate

the end reaction of glycogen breakdown. Although present as a

single locus in many salmonid species (Busack et al., 1979),

Alleridorf et al. (1977) showed a second locus is active in brown

trout but its poor resolution made it unreliable as a genetic

marker. Double banding activity has been reported in Atlantic

salmon (Cross and Ward, in press) but they feel this might be the

expression from a single locus. The second band of activity also

appeared in Tweed trout but not consistently and whether it is due to a second locus or is some secondary isozyme expression will have to be confirmed. 8 - a PGI - Genotypes

A - Common genotype

B - Variant allele at PGI-2 occurs infrequently

in samples from Tweed (less than 2%)

8 - b PGM - Genotypes

A - One banded PGM phenotype

B - Two banded PGM phenotype: significance

of second band is uncertain Figure 8 - Other Isozymes

Wit = -P01-2 - - P01-1

U

ii 8 - c SOD - Genotypes

Sample 4 on run 19 (MDH) shows the fast band for SOD: it is unclear if this is a genetic polymorphism.

This sample was of heart tissue of a brown trout smolt. Figure 8 - Other Isozymes

1, A .g

1 4 DISCUSION AND CONCLUSIONS

The use of isozyme data as an indicator of genetic variation provides a technique for integrating population genetics into fisheries resource management. The initial step is the description of genetically based protein variation. If genetic models can be reasonably verified then it should be possible to identify genotypes, analyse population structures and have some measure of the effect of man's activities. 4.1 ISOZYME VARIATION IN SCOTTISH FISH

In this study of isozyme variation in Scottish brown trout most of the polymorphisms recently reported in Scandinavian and Irish fish are noted.

- LDH-1(0) This variant originally designated as a fast allele appears in Scottish fish, though a great variability of isozyme expression was noted. It will be interesting to see from further biochemical genetic work what the nature of the variation at this particular isoenzyme is and if it has any evolutionary significance.

- LDH-5(105) This variant appears only rarely, in the east coast Scottish samples.

- AGP-2(50) This polymorphism occurs and shows significant differences between some of the sites.

- CPK-1(115) This polymorphism appears rarely or not at all in the four sites sampled. But frequency information from this isozyme should be treated warily as this allele is hidden when in the heterozygous state.

- MDH-3,4(125) & (80) Both these polymorphisms are indicated in the Scottish fish samples. They did not resolve well in the A- (Ridgway) buffer after Allendorf and Utter, 1978; but came out quite well on the B- (amine-citrate) buffer.

- MDH-2(152) This appears quite polymorphic in samples from the Tweed.

- MDH-1(0) This polymorphism might occur in

Scottish fish though a more carefull assessment needs to be made.

- ME-3 There appears to be some sort of isozyme variation at this locus though whether it has a valid genetic basis or is an artifact needs further investigation.

- ME-1 & ME-2 No variation was observed.

- PGI This enzyme resolves quite well and the

140 allele at PGI-2 (reported by Ferguson, 1980) also appears in

Scottish fish. The PGI-3 (110) alleleomorph )f Scandinavian fish was not.detected.

- PGM No genetic based polymorhism has been reported in brown trout. A second band of isozyme activity needs to be clarified as to its nature.

- ADH & 6PGDH Both these enzymes appeared as invariant bands.

- IDH Variation appeared in this enzyme though banding patterns were very bunched and difficult to interpret. A different more alkaline buffer might be more productive.

SDH This enzyme system did not resolve at all well.

- SOD A fast band of isozyme activity was observed in one sample. If this variant was a fast homozygote as it appeared to be, then its appearance as one in 75 common.homozygotes, with no heterozygotes, makes it a highly improbable event. Thus its genetic basis must be questioned.

Comprehensive surveys of isozymes will require a larger array of buffers, not all the systems attempted could be adequately resolved on the two buffer systems used in this study.

85 4.2 POPULATION STRUCTURES

Once the descriptive work and allelic frequency information has been gathered it is possible to structure populations based on significant frequency differences, departures from expected genotypic distributions and, if the data are sufficient, using indices of genetic separation. In their extensive work on

Scandinavian trout Ryman and Stahl (in press) have constructed elaborate dendrograms based on Nei's (1972) gene identity (I) that confirm the trend observed, in Pacific salmonids (Utter et al., in press) that population structuring is primarily geographic. Closely located populations cluster together in their genetic similarities; divergence is based, with some exceptions, on geographic seperation.

Ryman and Stahl (in press) have also noted a marked genetic heterogeneity in some river and lake systems based on significant frequency differences at the CPK-1 and AGP-2 loci. Genetic diversity was noted from 15 sampling sites on the River Storan and

11 sites on Lake Lulejaure and tributaries. This diversity was interpreted as signifying quite fine population subdivisions within river and lake systems. Together with evidence from Lake

Bunnersjoarna, where the two different LDH-1 alleles are fixed in apparently reproductively isolated demes (3.1.3) and in Lough Melvin where subdivisions appear to be based on trout phenotypes (3.1.1), there is some confirmation of Ricker's hypothesis that many stocks may inhabit the same lake or river system.

The isozyme data from the Scottish fish did not warrant using

W. genetic identity coefficients or dendrogram plotting. The two smolt sites (TW & NE) showed identical allelic frequency figures at AGP-2

(table-5). The Lewis fish (SC) showed a significantly lower frequency of the common AGP-2 allele. Unfortunately not enough data points are available to determine if any trends are emerging, such as consistent east coast I west coast frequency dichotomies, or consistency of phenotype expression (similarities of the two smolt sites).

Ferguson (pers. comm.) has suggested that a significant difference of the LDH-5(105) frequency between east and west coast

Irish fish may exist. This polymorphism occurs very rarely in the east coast Irish trout (similar to the frequency levels found in

Scottish east coast trout -table-3), but appears in higher frequencies on Ireland's west coast. Evidence may emerge soon from

Northern Ireland that will point to major structuring between an east coast trout and a western form (sewen).

It was not possible to subdivide the two river samples (1W) and

(NE) because of numbers and methods of capture. In both cases though, genetic homogeneity was implied by very close agreement to expected (Hardy-Weinberg) distribution of genotypes at the AGP-2 locus (table-5). Similarly the MDH-2 sample of the Tweed also conforms very well with expected genotype distributions (table-7).

The test of the significance of observed genotype distributions to Hardy-Weinberg expectations may not be a powerful enough test for the hypothesis of genetic homogeneity and panmixia however. The sampling may be too coarse to pick up fine subdivisions.

Subdivisions may occur that have similar allelic frequencies at the loci tested.

Genetic models of isozyme variation may not be valid.

These and other factors (Fairbairn and Roff, 1980) allow a reasonable probability of a Type II error to occur; that is we do not reject the hypothesis of homogeneous panmictic populations even when it is false.

If we allow that no statistical error has occurred, there may be a fairly good reason why brown trout from the Tweed and North Esk are genetically homogeneous while those of the Swedish River Storan are genetically heterogeneous. The Storan is composed of a series of lakes and within the 15 sample sites are 3 waterfalls that could effectively prevent migration (Ryman and Stahl, in press). The lakes alternating with a fast flowing river also provide a fairly heterogeneous environment. Localized population structures could develop that would be reinforced by drift and natural selection.

The trout samples from the Tweed were from several up-river burns and migrating smolt from down-river seine netting. Most if not all of these samples are likely to be progeny of migrating sea trout and homing to exact natal spawning tributaries is not likely to be as effective in fish that have to travel long distances. Localized population structuring may be less evident in tributaries accessible to migratory fish. There is also less environmental diversity in these Scottish rivers. It must be emphasized however that far more detailed sampling is necessary to corroborate the hypothesis of, genetic homogeneity. !_3 SYSTEMATICS

Questions arise as to the significance of sympatric discrete

stocks: Do the demes of Lake Bunnersjoarna represent evolutionarily

divergent populations, or do they represent short time fluctuations

in gene frequencies caused by population bottlenecks? Do the

phenotypes of Laugh Melvin and other lakes and rivers of Ireland and

Scotland represent evolutionarily divergent phenotypes, or do they just exhibit some ecological differentiation which is reinforced to

some extent by preferential mating for their "ecotype"? Isozyme

data can go a long way to answering some of these questions but far more material will have to be gathered.

Speciation and systematics in lakes has long been a matter of controversy and debate. In northern lakes the highly complex

interrelationships within the coregonids has fueled much , of this controversy. In his comprehensive review of speciation in the coregonids, Svardson (1979) emphasizes the importance of the glacial

events in the northern lakes. Repeated isolation, reinvasion and

introgression, and competitive and ecological forces, mould this group of fish into an elaborate sympatric species complex. Ferguson

(1980), influenced by his work on Irish coregonids (Ferguson and

Svardson, 1978), feels that a similar model of isolation and ensuing

introgression is responsible for the polytypic forms of brown trout.

The genetic divergence in brown trout has not been as great as it

has in the coregonids, and after reinvasion and the swamping process

"the ecological characters of the two populations are the last to disappear, since they are selected for persistance" (Svardson, 1979). Whether there is any evolutionary divergence between anadromous sea trout and river resident brown trout awaits further more substantial investigations. A great deal of isozyme data has been gathered on steel head (anadromous rainbow) and rainbow trout on the

North American Pacific Coast and no degree of genetic divergence has been observed from these data (Allendorf and Utter, 1979; Utter et al., in press). In my samples no remarkable isozyme differences were noted between the smolt samples (1W and NE) and brown trout samples (BR and SC) that are not indicative of simple geographic separation.

The Swedish work has dealt mainly with lacustrine stocks, but in some of the anadromous Swedish hatchery stocks tested, no great genetic divergence was apparent. Referring to this and the American work on rainbow trout, Ryman and Stahl (in press) express their opinion that anadromous behaviour may have evolved rather recently and that behavioural differences may not reflect large amounts of genetic divergence.

The view expressed by Ryman and Stahl (above) and by Ferguson

(1980) that brown trout phenotypes show evolutionary divergence is in contrast to the view of Kosswig (1973) (section 1.1.3) that these phenotypes are ecological modifications of the wide reaction norm of the genotype. At least with freshwater resident brown trout and sea trout; the ecological work of Campbell (1972) on the Tweed and of

Beau (pers. comm.) in the Kerguelens would indicate that it is often very difficult to differentiate these two forms and that they most likely arise from the same population. Splitting its phenotypic expression might be an evolutionarily wise investment for a species like brown trout.

Baker (1978) in his book on animal migration states that for territorial animals there is a cost incurred by migration; moving constantly downstream into conflict situations. Salmonids offset this cost considerably by forsaking territorial behaviour during their migratory phases and inclining towards shoaling behaviour

(Stuart, 1957). Hoar (1976) has noted that the more anadromous the salmonid species, the more marked is its shoaling behaviour. Other costs though are associated with migration; moving into unfamiliar territory leaves the animal more vulnerable to predation, and in obligatorily anadromous species, glacial rebound, with creation of barriers, leaves large areas inaccessible as habitat or spawning grounds. Living a sedentary life also has its costs; on the edge of a constantly fluctuating ice cap, it is not wise to invest wholly in a sedentary life form. Therefore it is evolutionarily advantageous for a species like brown trout to be able, within its genome, to invest in a dual life style and exhibit a "fixed heterozygosity" towards migration.

Duality of expression seems to be a feature of many characteristics in salmonids. Johnson (1972) has noted the phenomenon of bimodal body size distributions in salmonid communities. Ryman and Stahl (1979) in referring to this work hypothesized that genetic heterogeneity, as observed in the Lake

Bunnersjoarna populations, is the most likely cause of this bimodality. Thorpe (1977) in controlled environment rearing

91 experiments showed that bimodality of size distributions among

sibling populations of juvenile Atlantic salmon appears to be the norm. The analysis of variance for this distribution showed, that although modified by the environment, the major influence was genetic. Thorpe implies that this bimodality is the precursor of the smolting year classes that have been reported for natural

populations of both Atlantic salmon and brown trout (Went, 1962;

Harris, 1971). The evolutionary advantage of splitting smolting year classes can be argued for these species that live in arctic and north temperate conditions, where escape to sea may be influenced by climatic vagaries. Phenotypic dichotomies, even in relation to

size, do not necessarily imply genetic heterogeneity as hypothesized by Ryman and Stahl.

The evidence from the ecological work and some of the scant isozyme data would tend to support Kosswig's view of the nature of the difference between brown trout and sea trout. However

Ferguson's work, although so far confined to only one lake, does indicate initial evidence of, if not evolutionary divergence, then genetic divergence between some of the lacustrine phenotypes.

4.3.1 Interspecies Systematics

In at least two loci, LDH-5 and CPK-1, rare alleles that appear

in brown trout, migrate at the same rate as the common Atlantic salmon alleles (3.1.1 and 3.2.2). Likewise Khanna et al. (1975) have reported variants at these loci in Atlantic salmon that migrate at the same rate as the common trout alleles. It comes to mind that

92 this could indicate a certain level of introgression or persistent

"gene leakage" between these two species. It is also interesting to note that it is the variant LDH-5 allele that appears in such high frequencies in ferox trout. Although the low levels of these two variant alleles in the Scottish data (tables 3•& 6) lend support to this hypothesis, the high frequencies observed in some of the Irish and Scandinavian populations discount it. The sharing of alleles at these two loci most likely indicates fairly recent divergence between these two species.

Unlike most animals, introgressive hybridization does occur in certain groups of fishes (Hubbs, 1961). Payne, Child, and Forrest

(1972) reported that up to 1% of salmon and grilse caught in north east British waters might be F-i trout hybrids. These authors felt that no further introyression, other than the occasional F-i fish, would occur due to the genetic instability of further back-crosses and the extreme unlikelihood of F-2 crosses (P< 0.000005). Studies of back-crosses of F-i individuals to salmon and trout (Nygren et al., 1975) confirm the inviability of these crosses. Only six

individuals were left by the following August of back-crosses to

salmon and none to back-crosses with trout compared to 1589 and 1685

individuals left in control groups. Many salmonid species can form

F-i hybrids with closely related species but most go no further than this. After massive hybridization of pink and chum salmon, where nearly half a million F-i hybrids were released into a fishery,

subsequent electrophoretic analysis (May, Utter and. Allendorf, 1975;

May, 1975) showed no permanent introgression.

93 Permanent introgression may occur between some species. Behnke

(1970) and Behnke and Zarn (1976) have reported that whereas rainbow trout and cutthroat trout live sympatrically in interior Pacific coast streams without significant hybridization, introductions of hatchery rainbows can often produce hybrid swarms and many unique cutthroat trout demes have been swamped in this way. The complex array of the Salmo group in Western North America has been much disrupted by man's activities and many of the unique species of this region are endangered with swamping by introduced rainbow trout

(Behnke and Zarn, 1976)

94 4.4 MAN'S INFLUENCE ON FISH STOCKS

Man's influence on the genetic identity of salmonid species is undoubtedly great, perhaps on the same scale as the glacial events of the past. The disruption of the environment and the interruption of ecological differences can cause the delicate balance between closely related species to be interrupted and thus cause the introgression and swamping observed by Behnke. Most species have diverged far enough that this is not so much of a problem, though it is a concern where genetically distinct demes within a species have evolved and coexist.

Plantings and the use of hatchery fish are an important and necessary part of fisheries management. Although the facilities offered by hatcheries are needed to maintain salmonid stocks, concern is being expressed about possible loss of genetic diversity with their use.

Allendorf and Utter (1978) looked at the genetic variability in

41 populations of native and hatchery rainbow trout. No significant loss of heterozygosity could be seen in any of 12 hatchery anadromous populations and only one out of 8 hatchery resident populations showed any significant loss of heterozygosity _(30 gene loci were used). This population was in the University of

Washington hatchery where over 40 years of intensive artificial selection work has been carried out. Allendorf and Utter blame this artificial selection and inbreeding for the apparent loss of genetic variability in this population. In four strains of California

95 rainbow trout whichhave undergone severe inbreeding and artificial selection Busaketal. (1979) using 24 gene loci on 1462 fish found no significant loss of heterozygosity from wild strains. These authors concluded genetic diversity was maintained from the initial high heterozygosity of rainbow trout by the mixing of sources in establishing these lines and perhaps by balancing selection favouring heterozygotes within the hatchery programme. These two studies form contradictory conclusions about the loss of genetic variability associated with high artificial selection and inbreeding. Without details about the levels of inbreeding

(inbreeding coefficient F -appendix 2), we would have to conclude that as only one out of the 24 hatchery populations used in these studies shows any significant loss of heterozygosity from wild stocks, normal levels can be maintained, at least in reasonable regimes of inbreeding and artificial selection.

In their work on Scandinavian hatchery brown trout Ryman and

Stahl (1980) found a loss of variability. Although this study only looked at 2 loci (AGP-2 and CPK-1), these authors felt that significant allelic frequency shifts and loss of heterozygosity. took place. They implicate a poorly managed breeding programme at the hatcheries which does not take into account a reasonable "effective" number of breeding parents, especially the practise of relying on too few males. They also noted that the inbreeding depression

indicated by the proteins also showed up in the general health and

physical characteristics of the fish. If Ryman and Stahl's conclusions about the loss of variability are valid (only 2 loci are used and some of the small, sample sizes 12, 20 & 25, make it

M. difficult to get accurate frequency figures, especially for CPK-.1), then a different picture is presented to that of rainbow trout.

This could be due to the inherent differences between the two species.

Perhaps the balancing selection hypothesized by Busak et al. (1979) works over 24 loci but is not effective on AGP-2 and

CPK-1.

The establishment of rainbow trout lines from mixed backgrounds may give a far higher initial heterozygosity figure than locally established brown trout hatchery populations.

Inbreeding may be much more severe in Swedish than in

American hatcheries (no figures on inbreeding coefficients F are

available in any of these papers for comparisons).

More work needs to be done on the possible loss of genetic variabilty in hatchery progammes.

The influence of man's activities and his hatchery programme on

fish populations is not always noticed as shifts or changes in

isozyme frequencies. Ecological adaptions have been selected for in

natural stocks but this selection has been relaxed in hatchery

stocks and sudden influxes of hatchery fish can upset ecological

balances. The disruption of ecological barriers to hybridization as

implicated in the introgression of some of the cutthroat trout

populations by hatchery rainbows (Behnke and Zarn, 1976 and above), may be more important than changes in isozyme frequencies. These

authors claim that some unique populations of cutthroat trout have

been lost in this way. Reisenbichler and McIntyre (1977) have noted

that hatchery steel head trout when interbred with wild trout may

97 reduce the number of smolt produced. Krueger and Menzel (1979) have demonstrated how maintenance stocking of hatchery fish on wild brook trout has altered the native gene frequency distribution; and they have speculated that this change is not brought about by interbreeding but by some selective feature of the interaction between the two stocks. 4.5 CONCLUSIONS

The alterations on the nature of fish stocks, as with genetic changes due to selection and inbreeding in hatchery stocks are not always related to change and loss of isozyme frequencies. This points to one of the weaknesses of using isozyme data in representing genetic variation. Isozyme data is based on structural proteins of enzymatic function many of which are involved in glycolytic pathways; thus it does not represent a truly fair and unbiased part of the genome. This bias can particularly effect our measures of genetic diversity and it means some of the formal methods of quantifying this diversity such as using average heterozygosity (H) and genetic identity formulas (I) need to be approached cautiously. But the limitations of isozyme data does not invalidate it. It provides one of, the few practical ways of identifying genotypes and looking at the genetic structures of natural populations. The effectiveness of using isozyme data requires;

- many and varied loci,

- good sample sizes in excess of 100 or more if possible,

sampling from many data points,

- controlled breeding if possible.

If our assumptions of the genetic basis to isozyme variation are reliable, then the analyses of the data becomes a statistical exercise. Too many of our present genetic models are questionable however, and far more data will have to be gathered to offer Hatchery Programmes

Ryman and Stahl's (1980) study of the loss of genetic variability in hatchery populations is in contrast to the evidence presented in more detailed studies of rainbow trout (Busak et. al., 1979).

Although it is difficult to compare these two studies; they are two different species and no figures are given to compare management practices and inbreeding coefficients (fl.

The deleterious effects of inbreeding can be found in hatchery populations (Kincaid, 1976 and Dodge, pers. comm.), though at what level this begins to occur and how effective isozyme variation is as an indicator of severe inbreeding needs to be determined. It is perhaps best to adopt a cautious approach here and accept the recommendations of Ryman and Stahl (1980) that high numbers of effectve parents be kept; at the very minimum 30 males and 30 females for a brood stock.

The ecological inadaptiveness of hatchery fish and the disruption of normal ecological balances in natural populations can be one very important influence of hatchery stocking. This aspect of our hatchery programmes should be explored much further.

Evolutionary Divergence and Genetic Heterogeneity

The conclusions of many of the population geneticists appears to be that there is a lot of evolutionary divergence in brown trout

(the two demes of Lake Bunnersjoarna and the phenotypes of Lough

100 Melvin). This conclusion needs to be approached cautiously, genetic

heterogeneity need not imply evolutionary divergence. There is a good deal of evidence that many of these protein polymorphisms are

adaptively neutral (1.4.1). In small spawning streams random

fluctuations in gene frequencies may easily occur even when these

populations normally live sympatrically in the same lake or river

(4.2). This kind of heterogeneity has been noted in some of the

Swedish rivers and lakes (4.2). The Lake Bunnersjoarna demes may be

a more extreme example of this genetic heterogeneity where a variant allele for LDH-1 has become fixed in one of the populations (3.1.3).

The division of the trout phenotypes of Lough Melvin into discrete populations is based on the assumption that agreement to

H.W.E. distributions implies genetic homogeneity within these discrete groups. Although there are some shortcomings to using this as a test for genetic homogeneity (4.2), the, sharp difference in allelic frequencies at LDH-5 does imply some genetic difference.

More evidence needs to be gathered especially from other lakes.

Ryman and Stahl (4.2) in quite detailed studies have shown a good deal of genetic heterogeneity, at least in fresh water resident river and lake dwelling brown trout. This localized population structuring may be less apparent in more migratory fish (4.2). This conãlusion is based on the apparent homogeneity of samples that arise from many and unknown spawning tributaries of the Tweed and

North Esk; more extensive sampling is required to corraborate this hypothesis.

101 It is hard to assess how effective natural selection is on these protein polymorphisms. Certainly the geographically based genetic heterogeneity observed (4.2) would tend to support the idea (1.4.1) that they are neutral. On the other hand there is some evidence that the polymorphism for LDH-4 in rainbow trout has some adaptive significance (3.1.2) and Busaketal. (1979) have suggested that protein heterozygosity may be actively selected for under strong regimes of artificial selection in hatcheries (4.4). In hatchery management it is probably safest to assume that loss of variability or severe shifts in frequencies may be harmful to fish stocks and they should be maintained to natural levels of frequency distributions.

The information that is presently available should provide the groundwork for the understanding of population structuring in brown trout. It is hoped that the data presented in thisthesis can add to this ground work. A lot more data will have to be gathered however, before a clear idea about the genetic nature of brown trout and its natural population structures can emerge.

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117 ADDNnTY 1

GLOSSARY (from May, (1975); King, (1974))

Allele: one of several alternate forms of a gene. Autosome: a chromosome other than a sex chromosome. Balancing selection: selection favouring heterozygotes. Codominant: both alleles of a pair are fully expressed in the heterozygote. Dimer: active protein (enzyme) composed of two polypeptide chains. Diploid: pertaining to cells or organisms having two sets of chromosomes. Disomic: having one chromosome in the complement represented two times in each nucleus. Ecotype: a race (within a species) genetically adapted to a certain environment. Fixed heterozygosity: heterozygosity from the two alleles of a duplicated loci pair. Founder principle: newly isolated population will diverge from parent population due to sampling errors and different evolutionary pressures.. Gene: a unit of genetic inheritance. Generally, a gene refers to the part of the DNA molecule that encodes a single enzyme or structural protein unit. Genetic drift: change in allele frequency due to random occurrences in a population. Genome: the entire genetic complement of an individual. Genotype: all the genetic characteristics that determine the structure and functioning of an organism; often applied to a single gene locus to distinguish one allele, or combination of alleles, from another. Haploid: pertaining to cells or organisms having one set of chromosomes. Heterodimer: a dimer composed of two different subunits. Heterotetramer: a tetramer composed of two to four different subunits. Heterosis: hybrid vigour. Heterozygous: containing two forms (alleles) at a gene locus. Homodimer: a dimer composed of identical subunits. Homotetrarner: a tetramer composed of identical subunits. Homozygous: containing two identical alleles at a gene locus.

118 APPENDIX 1 (continued)

Gene pool: the total genetic information possessed by the reproductive members of a population. Gluceonogenesis: glucose synthesis from lactate/pyruvate precursors. Inbreeding: the crossing of closely related individuals. Introgression: incorporation of genes of one species into the gene pool of another species. Karyotype: set of characteristic chromosomes of a particular species. Linkage: association in inheritance of genes because of proximity on the chromosome. Locus (plural, loci): The position that a gene occupies on a chromosome. Mendelian segregation (inheritance): separation into different gametes and then into different offspring of the two genes at a single locus. Monomer: active protein (enzyme) composed of a single polypeptide chain. Mutation: any change in the genotype of an organism occurring at the gene, chromosome, or genome level; usually applied to changes in genes to new allelic forms. Ontogenetic: development of individual from fertilized egg to adult organism. Panmictic: population in which mating is random. Phenotype: physical expression of the interaction of genotype and - the environment. The banding pattern on the starch gel is one form of the phenotype of an individual. Polymorphism: occurrence of more than one allele at a locus in a species. Polypeptide: a molecule made up of amino acids linked together by peptide bonds. Polytypic: species that has a number of specialized races. Regulatory: (pertaining to enzyme synthesis) affecting its level of synthesis. Segregation: separation of, the genes at a locus derived from the male and 'female parent into different gametes during meiosis.

Segregation load: the genetic disability in a population due to the segregation of unfit homozygotes from advantageous heterozygotes. Stochastic: process involving a series of random steps.

Structural gene loci: specifies formation of polypeptide chain.

Tetramer: active enzyme composed of four polypeptides.

Tetraploid: pertaining to cells or organisms having four sets of chromosomes.

119 APPENDIX 1 (continued)

Tetrasomic: having one chromosome in the complement represented four times in each nucleus. Transcription: the formation of a similarly sequenced messenger RNA from a specific section of DNA (a gene). Translation: the formation of a polypeptide chain of amino acids on a ribosome, directed by a specific messenger RNA molecule.

120 APPENDIX 2

STATISTICAL ANALYSIS

from Nei, 1975; Dobzhansky et al. 1977; Ferguson, 1980

Calculation of Allelic Frequencies

The frequency of an allele is given by

2s +11 0 C 21v

where H0 = number of homozygotes for that allele H = number of heterozygotes for that allele Iv = number of individuals examined

The 'standard error' of the frequency of an allele (p) is estimated by

• /(i-.) \/2N

Heterozygosi ty

The calculated heterozygosity per locus is

H= 1 - Ex 2 L 1-

where x. is the frequency of the ith allele at a locus.

The heterozygosity of a population can be expressed as the average frequency of heterozygous loci per individual (H.). This is estimated by averaging. (over all individuals) the proportion of heterozygous loci observed in each individual.

121 APPENDIX 2 (continued)

Hardy-Weinberg distribution

If a population is in Hardy-Weinberg equilibrium, then the frequencies of genotypes will be in the ratio of p 2 , 2pq and q 2 , for a two-allele polymorphism, where p is the frequency of allele A and q is the frequency of allele B.

The difference between the observed and expected values can be tested for statistical significance, using a x2 test for goodness of fit. Alternatively, and preferably if the expected values are small, the log likelihood x2 test (G-test) can be used.

G = 2ZObsln I [JObs 't Exp

= 2EEObslnObs - (2.30259)EObs log ExpJ

The distribution of G will be approximated by the x2 distribution, and the significance of the value can be found in appropriate tables. (For further details of the G-test and its advantages see Sokal and Rohlf, 1969).

Genetic identity and distance

Nei's coefficient of genetic identity (I) between two taxa is given by

Exy 1= v'(E.x.2Ex.2 )

where x. and y are the frequencies of the ith allele in 'V 'V populations x and y respectively

I = 1 when x and y are monomorphic for the same allele, and = 0 when x and y are monomorphic for different alleles.

The mean genetic identity (.T) is the mean over all loci studied (including monomorphic ones) and is most conveniently calculated as

122 APPENDIX 2 (continued)

I xy .1(isy I

where I , I and I are the arithmetic means, over all loci of xy x y E,.y., Ex. 2 and Ey 2 respectively.

The genetic distance (D) is estimated by

D = -mI which can be obtained from tables of natural logorithms or on many electronic calculators.

The time of divergence (T.) of two taxa can be estimated by

T = 5xlO 6 D

Inbreeding Coefficient

F is the inbreeding coefficient is the effective size of the population. N

Loss in genetic variation due to inbreeding as a factor of small population size is calculated as

AF = ____ 2N e

A major factor which influences N is the proportion of each sex in the population

= 4NdN9 N e

Ryman and Stahl (1980) (Section 4.4) indite a highly inbalanced sex ration, thus low N in the loss of genetic variation observed in Swedish hatcheries.

123