THE GENETIC EFFECT OF STOCKING AND POPULATION STRUCTURE OF BROOK TROUT (SALVELINUS FONTINALIS) IN THE BEEF RIVER. A Thesis. Submitted to the Faculty

Transcription

1 THE GENETIC EFFECT OF STOCKING AND POPULATION STRUCTURE OF BROOK TROUT (SALVELINUS FONTINALIS) IN THE BEEF RIVER A Thesis Submitted to the Faculty of University of Wisconsin o - La Crosse La Crosse, Wisconsin by Kevi n Ca 11 en In Partial Fulfillment of the Requirements for the Degree of Master of Science in Biology August 1983

2 '~ c:v'' UNIVERSITY OF WISCONSIN - LA CROSSE La Crosse, Wisconsin COLLEGE OF ARTS, LETTERS AND SCIENCES Candidate: Kevin Callen We recommend acceptance of this thesis to the College of Arts, Letters, and Sciences in partial fulfillment of this candidate's requirements for the degree Master of Science in Biology. The candidate has completed his or her oral defense of the thesis. Thesis approved: O:Jkt7~rh ~~# / /j /JJ'3 Committee Chai rpersoj'f'-) Date ;> // ~~~/yr3 Date ' :' :'? /~, (/ 'r. #? t,j_,x~: f./.."-" '" /~;..j,5 Date 30 ~ /c;$'3 Oat.w (, /u:-.~ I 1 \~lc r.j N--t (j t \,~- SEPj. (" / 761 ean, Office of Graduate Studies Date 8,1 fagco

3 i i ABSTRACT The management of trout populations in streams often includes stocking hatchery-reared fish to supplement natural reproduction. Due to artificial selective pressures, hatchery populations may vary genetically from wild fish. In the relatively stable hatchery environment, the conditions are conducive to rapid genetic shifts which may be preserved for future use in the form of brood stock. The hypothesis of this study is that stocking has an impact upon the genetic makeup of the wild populations of trout. By examining commonly accepted genetic traits in the hatchery trout and different subpopulations of wild trout, an assessment was made concerning the impact of stocking upon the genetics of the wild trout. Trout were collected from seven stream sections in the North and South Fork of the Beef River and from the St. Croix State Fish Hatchery. Whole eye and liver samples were analyzed for six enzymes using starch gel electrophoresis. Genetic polymorphism was seen in four of the six enzyme systems examined. Distinct genetic divergence between stream sections identified local populations of trout within the North and South Fork of the Beef River. Differences in natural selective pressures, associated.. with location between and within the drainage basin, may account for the genetic variation in the wild trout. Homogeneity between stream sections stocked for 22 years and stream sections stocked for 3 years may be due to the effects of migration. The St. Croix Hatchery tish represent a genetically distinct population in comparison to the genetics of the wild trout. No interbreeding between hatchery trout and wild trout was evident, but a behavioral/ecological interaction may account for the genetic divergence in heavily stocked stream sections. The interaction was identified by an increase in heterozygous individuals in heavily stocked stream sections. The increase in heterozygotes was not in excess of Hardy-Weinburg equilibrium, but indicated a trend in heterozygosity. The possibility exists of managing trout on a stream section to stream section basis. Stream improvement. particularly in stream sections designated as stocking sections, would decrease migration ~ increasing the environmental heterogeneity. Evidence for genetic divergence between the St. Croix Hatchery trout and the wild trout signifies a need for the preservation of native gene pools in the.. ef River.

4 iv TABLE OF CONTENTS LIST OF TABLES. v PAGE LIST OF FIGURES vii INTRODUCTI ON.. 1 Hy pot heses Objectives Study Site Descri pt ion 2 Previ ous and Related Research METHODS 12 Field Collections 12 Gel Preparation 15 Statistical Analysis 21 RESULTS 22 Variant Enzymes 22 Lactate dehy drogenase 22 Isocitrate dehydrogenase 24 Hexose-6-phosphate dehydrogenase 27 Hardy-Weinburg Equilibrium 33 Genetic Comparison of Hatchery and Field Collections 34 Within-stream variation 37 Between-stream variation 38 Genetic Distances Between Stream Sections. 42 Genetic similarity between stream sections 46 Relations with the genetics of the Beef River brook trout. 50 Levels of Population Structure. 53 DISCUSSION ~ Hardy-Weinburg Equilibrium. 57 Population identification 58 Beef River brook trout population 58 Hatchery brook trout popul at ions 59 Relationship Within Genetically Defined Populations 61 Stocking Drainage position 62 Ecological and behavior interactions between the hatchery and wild trout 63 Management Recommendations 64 LITERATURE CITED. 66

5 v TABLE LIST OF TABLES PAGE 1. Stocking data of stream sections in the Beef River according to years of stocking, type of trout, and stocking rank Buffer systems and their potential used in electrophoresis A. Enzyme, tissue, buffer system, and stain solutions used for electrophoresis LOH-3 phenotypes and allelic frequencies with Hardy- Weinburg equilibrium probability values. 4. IDH-4 phenotypes and allelic frequencies with Hardy- Weinburg equilibrium probability values 5. IOH-3 phenotypes and allelic frequencies with Hardy- Weinburg probability values 6.H6PO-l phenotypes and allelic frequencies with Hardy- Wei nburg probabil ity val ues Chi 2 test of homogeneity with the St. Croix Hatchery using IDH-4 phenotypic frequencies Chi 2 test of homogeneity with the Osceola population using LDH-3 phenotypic frequencies Chi 2 test of homogeneity with the St. Croix Hatchery using LOH-3 phenotypic frequencies CHi 2 test of homogeneity with the St. Croix Hatchery using H6PD-l phenotypic frequencies Chi 2 homogeneity testing at the LOH-3 and IDH-4 locus withi n st ream sections Chi 2 values and probability values for homogeneity hypothesis at the LOH-3 locus in stream section to stream section Chi 2 values and probability values for homogeneity hypothesis at IOH-4 locus in stream section to stream section

6 vi TABLE LI ST OF TABLES 14. Genetic similarity matrix based in gene frequencies using Nei"s index of genetic similarity Rank correlation analysis of drainage position and IDH-4 allelic frequency using 0 and 100. r = Spearman rank correlation coefficient. s Allele frequencies and F-statistics calculated from the observed phenotypes. Group A includes samples in Jackson and Trempealeau Counties. Group G samples are from Trempealeau County and Group C samples are from Jackson Cou nty 56 PAGE 49

7 vii FIGURE LIST OF FIGURES PAGE 1. North and South Fork of the Beef River and sample sites 3 2. G-6-PD and MDH phenotypes in brook trout eye homogenate samples LDH-3 phenotypes in brook trout eye homogenate samples IDH phenotypes in brook trout liver samples H6PD phenoty pes in brook trout 1i ver samples Percent frequency of IDH-4 variation within three equidistant stream sections in the North Fork of the Beef River Percent frequency of LDH-3 variation within three equidistant stream sections in the North Fork of the Beef River. ~ Percent frequency of LDH-3 variation between North and South Forks of the Beef River Percent frequency of IDH-4 variation between North and South Forks of the Beef River. 43 Cluster analysis of trout at LDH-3 stream sections and St. Croix Hatchery Cluster analysis of stream sections and St. Croix Hatchery trout at IDH-4 48 Stream sections in relationship to percent frequency heterozy gos ity and stock i ng rank... of 47 52

8 INTRODUCTION Hypotheses Brook trout t Salvelinus fontinalis t have been stocked in Wisconsin streams for more than fifty years. Up until 1975 t all the brook trout stocked originated from the Osceola State.Fish Hatchery. An inbred population selected for color t shape t and growth has been maintained at this hatchery. This strain of fish was eliminated from the stocking program in 1975 due to a progressive infestation of furunculosis disease. In April of 1975 a New Hampshire strain was introduced to Wisconsin streams to replace the diseased strain. The origin of this strain is Nausha t New Hampshire. This inbred population selected for body conformity-coloration and egg production has been maintained at the St. Croix Falls Fish Hatchery located in St. Croix Falls t Wisconsin. Using previous electrophoretic data supplied by Krueger (1976) on the Osceola strain t and ny own data on the New Hampshire strain t a comparison of genetic variability between the two strains was made. Also t a comparison of the hatchery fish with the field populations in terms of genetic characterization was determined. The hypothesis ofule study \'/as that stocking had an impact on the genetic makeup of the native populations of trout. By examining commonly accepted genetic traits in. the hatchery fish and different subpopulations of native fish an assessment was made concerning the impact of stocking upon the genetics of the native trout.

9 2 Objectives The goal of this study wasto define the population structure of brook trout in a Wisconsin stream. It was designed to learn more about the genetic impact of stocking hatche~ trout on a native brook trout population. The research objectives are as follows: 1) to provide an estimate of the geographic differentiation of brook trout defined genetically ~ electrophoretic data; 2) determine the spatial relationships of breeding units among native brook trout; and 3) to determine if stocking hatchery trout has had any genetic impact upon the native trout population. Study Site Oescri pt ion In order for this investigation to be useful as a fisheries management tool, certain criteria concerning the study area have to be met. The stream chosen had to be representative of all brook trout fisheries in Wisconsin. The study area also had to possess a varied stocking history. The Beef River in Trempealeau-Jackson County fulfills the requirements for this study. Brook trout, Salvelinus fontinalis, are indigenous to this region (MacCrimmon and Campbell 1969) and thus, a comparison between locally adapted brook trout and hatchery trout genotypes can be examined. The Beef River is a clear, soft water stream that flows in a general westerly direction (Figure 1). The headwaters of the Beef River originate in Jackson County and flow through Trempealeau County and finally enter the Mississippi River in Buffalo County.

10 o II'" aca'. 2 SOUTH FORK Figure 1. North and South Fork of the Beef River and sample sites. w

11 4 The study area starts where the Beef River branches to form two second order streams. The North and South Fork are formed from the branching of the Beef River at Osseo, Wisconsin in Trempealeau County. Up until 1975, only the upper 4.8 km of the North Fork was considered class I. The remaining 9.0 km was class II. The entire South Fork was considered class II trout waters. Presently, the Trempealeau-Jackson County line seperates class II from class I trout water. Previous and Related ReseQrch The use of stocking as a method to maintain an exploited fishery has been a practice for years. Recently, various stresses have been placed upon fisheries in Wisconsin and other states by processes such as agricultural runoff, point source pollution, overfishing, and the introduction of exotic fish. Stocking has been used as a management tool to rehabilitate fish populations. The salmonids have been used for stocking because they are easily cultured and preferred by anglers. Presently, the number of trout stocked in the United States exceeds 70 million annually including approximately 800,000 into Wisconsin inland waters (Moring 1982~ Klingbil, personal communication). Hatcheries are the supplier of fish to sustain a stocking program. The hatchery facilities produce fertilized eggs, fry, fingerling and catchable trout as a program of maintenance stocking. The development of the cultured stock is through artificial selection which operates independently of natural environment (Smith 1961). In the relatively

12 5 stable environment, the conditions are conducive to rapid genetic shifts which may be preserved for future use in the form of brood stock (Shuck 1948; Calaprice 1969). There is a risk involved in the development of a particular strain of fish. The selection of brood stock with the subsequent production of large numbers of offspring from few parents leads to genetic deterioration resulting from inbreeding (Ryman and Stahl 1980; Kincaid 1976). The literature contains experimental evidence of gene arrangements that are advantageous in one environment, but neutral or deleterious in another environment (Dobzhansky 1951; Ayala 1968). This may lead to problems for the hatchery manager. Genetically, the brood stock is a product of the hatchery environment and the selection regime of the hatchery manager. Selection for characteristics of rapid growth, body conformity.cqj or~t ion,.. egg.. P!:Q9u~t i on and di sease res i stance have hi stori ca lly been associated with standard hatchery practices (Donaldson and Olsen 1955; Smith 1961; Nelson, personal communication)..-'...'~~.~.'.. ",,; The stocking of streams has been categorized into three classes depending on the stocking intensity (Thaemler 1975). A class I stream, or stream section, is not stocked due to its favorable habitat for natural reproduction. Class II streams have habitat suitable for trout, but spawning reproductive habitat is limited or lacking. Class III may be defined as a "put and take" fishery. Trout reproductive habitat does not exist, and therefore, fish are present only after stocking. A common practice is to stock legal-size trout in class III streams with the majority of these fishes being harvested shortly after planting. Compared to the Class III stream, Class II is a

13 "put, grow and take" situation and does show some carryover of planted trout. To supply the demand for trout to anglers, catchable trout are planted by state and federal fish management agencies. To a degree, the role of fish management is to adequately maintain the fish stock so the supply nearly reaches the demand. Recently, the effects of stocking hatchery trout on natural populations has led to discussion between fishery biologists and anglers. (Nicholas, Reisenbichler and McIntyne 1977). Stocking trout is commonly thought of as maintaining or improving yield with little effect upon existing native trout populations. Stocking is of concern to anglers in terms of providing fish for sport. However, knowledge of the population dynamics of a trout stream is necessary to justify a stocking program which must be the decision of the management agency concerned (Millard and MacCrimmon 1972). Population enumeration and the stocking of trout has been examined in numerous studies. Vincent (1974) found when hatchery-reared rainbow trout (Salmo gairdneri) were added to a self-sustained wild trout population, wild trout populations decreased. When stocking ceased, wild trout populations increased dramatically. Survivability of hatchery-reared trout has been compared to native and nativedomestic cross with the native showing superior survivability and native-domestic cross intermediate survivability (Miller 1954; Mason et ale 1967). In experiments conducted in the Adirondack Mountains of New York, wild strains of brook trout (Salvelinus fontinalis)

14 7 were superior to domestic strains in growth and survivability (Flick and Webster 1964). Fraser (1981) found that hybrid and wild brook trout were recovered at rates two to four times as great as the domestic strain. The study concluded that the domestic strain used in stocking programs in Ontario waters survived poorly after release. Webster and Flick (1981) concluded from their stu~ that rearing native wild strains to maturity in a hatchery, or domestic strains in a natural environment, did not affect the survival, suggesting the superior performance of the wild strain of brook trout was inherent. Several studies have revealed behavioral differences between hatchery-reared and wild salmonids, particularly Atlantic salmon. A study by Symons (1969) indicated that, unlike the juvenile wild salmon, the hatchery-reared salmon did not respond to overcrowding by normal downstream migration when planted in a stream. Dickson and MacCrimmon (1982) found a distinct difference in behavior between both hatchery-reared Atlantic salmon and wild salmon, and between hatchery-reared Atlantic salmon and wild brook trout. The authors indicated that the behavior of the Atlantic salmon was significantly altered when hatched and reared under hatchery conditions. So distinct, are the changes, the authors contended, that the hatchery fish behaved as a different species in their environmental demands and interactions with wild Atlantic salmon and wild brook trout. Pianka (1978) noted that as taxonomic distance decreased, environmental demand and aggression increased. The level of interaction varied depending upon stocking conspecifics or different species.

15 8 Ecological differences such as food habits, habitat preference, vulnerability to angling and vertical position within the water column have been reported for a number of salmonid species (Behnke 1972; Trojnar 1972; Allendorf et al. 1977). To manage salmonid communities solely on ecological data without an adequate description of the reproductive relationships within a group can be misleading (Johnson 1972). Ryman et al. (1970) indicated that ecological investigations are not valid unless based upon accurate descriptions of the reproductive relationships. Alteration of habitat and angling regulations within a trout fishery have been successful (Saunders and Smith 1962; Hunt 1971, Hunt 1981). The management of fauna based, on habitat requirements and administrative boundaries without considering the functional characteristics of the species is of concern (Smith et al. 1976; Ryman et al. 1977). The breeding unit is of importance when managing a community of potentially interbreeding individuals at a given locality. Given that distinct differences exist between the hatcheryreared and native fish, stocking may have an effect upon the locally adapted stock. Hqyt (1974) used meristic characters to determine the effects of stocking varieties of northern smallmouth bass (Micropterus dolomie~) on populations of Neosho bass. The stuqy suggested an alteration of values for meristic characters ensued in populations where stocking had occurred. The shift of characteristics was in the direction of the planted fish. Gard and Seequist (1965) also studied the effect of stocking using hatchery rainbow and cutthroat trout, (Salmo larki), on native rainbow populations. and meristic counts suggested a slight divergence from the native :'~.,r.1 r

16 9 rainbow populations. Morphometric and meristic counts suggested a slight divergence from the native rainbow trout. Problems arise when separating the morphological variation into genetic or environmental expressions. Barlow (1961) noted the possible environmental influence upon these characteristics which reduce their sensitivity for detection of genetic change. Lindsey (1981) noted that even relatively stable morphological characteristics, such as gill rakers in corogoninae fishes, responded quickly to changes in its environment. The author concluded that changes in morphology with time, or with distance, does not imply genetic differences. If temporal and spatial variability exists in native salmonid populations (Vaughan 1947; Burger 1964; Lewontin 1965; Raleigh 1967), identification of the gen~tic structure would be an important management resource. Gel electrophoresis of enzymes provides an effective tool for delineating the genetic diversity present within a species. Enzymes are polypeptide chains made up of a sequence of amino acids that are the product of the.sequence of the DNA nucleotides that comprise the structural gene. Most changes in the DNA base sequence will be reflected in changes in the sequence of amino acids. Many of these changes wi 11 affect the mobil ity of the enzyme mol ecul e in an electric field by altering its charge or shape. The degree of s~~aration on a gel depends on the molecular charge and size of the protein (Gnrd r)t1 1969~ Brewer 1970). Negatively-charged proteins migrate towards the positive pole of the apparatus and positive proteins to the negative pole. The electrophoretic separation of proteins, coupled with specific enzymatic stains, are an expression

17 10 of codominat alleles at individual loci. The interpretation of electrophoretic data combines Mendelian and population genetics with electrophoretic techniques. To verify the basis of electrophoretic polymorphisms, inheritance studies along with Mendelian principles are required to demonstrate the mode of inheritance (disomic or tetrasomic) for duplicated systems (Bailey et ale 1970; Stoneking 1979; Stoneking et ale 1981). The use of electrophoresis has provided insights into the breeding structure of natural populations and has led to the description of temporal and spatial relationships (de Ligny 1969; Allendorf and Utter 1979; Fairbain et ale 1981). Genetic studies using isozymes have made a contribution toward efforts to preserve genetic resources, locate differences between populations and maintain the integrity of distinct gene pools of fish. Allendorf and Phelp (1980) detected a reduction in genetic 1. " variation at isozyme loci in hatchery west-slope cutthroat, (Salmo clarki, lewisi) in comparison to the original wild stock from which it was derived. The native west-slope cutthroat trout population has undergone a reduction in numbers such that the hatchery cutthroat represents an important "gene bank" for the subspecies. The authors contend that habitat destruction and introgression of rainbow trout reduced the cutthroat trout populations in many of its native waters. Northcote et ale (1970) suggested that either introgressive hybridization or the introduction of new genetic material from a different species, may have influenced the allelic frequency at the LDH locus in native trout due to stocking of cutthroat. Eckroat (1971, 1973) noticed in populations of Pennsylvania brook trout that one population exhibited

18 11 a high frequency of a rare allele which was only observable in the hatchery population. Stocking was believed to be the reason for the occurrence of the rare allele. M~ller (1970) noted that stocking may have contributed to the genetic diversity in Atlantic salmon (Salmo salar) populations. The study concluded that genetically distinctgloups of Atlantic salmon exist, but introductions may have affected the gene frequencies of the original populations. The effect was an increase in heterozygosity at the transferring loci in the Atlantic salmon. Krueger and Menzel (1979) noted a significant correlation between LDH-B 2 allelic frequencies and stream stocking histories, with the wild type allele decreasing in importance as stocking intensity increased. They speculated that an alteration of selectlve pressures induced by ecological interactions between the hatchery and wild stocks caused the genetic variation. In 100 undisturbed populations of brown trout, Salmo trutta, Ryman and Stahl (1981) never found a si gnifi cant excess of heterozy gotes. The research suggests that crosses between genetically distinct populations are expected to result in an excess of heterozygotes. The authors stated that previously existing reproductive barriers between natural populations had broken down due to introductions and that forced matings had altered the genetically distinct natural populations.

19 12 METHODS Field Collections Brook trout were collected by electroshocking using a D.C. stream shocking unit and transporting in plastic buckets to the processing station where ice was added to the water to maintain a cool temperature. A total of 177 brook trout were collected, including 54 fish from the St. Croix hatchery. To assure that stream sampled fish were not from recent hatchery plantings, only fish less than 15 cm. long were collected. Though the sampling effort was equal, 10 to 39 trout were taken per sample. Differences in stocking intensity on various stream sections of the Beef River (North and South Fork) were evaluated by a ranking procedure. Stocking in specific stream sections has been documented by the Department of Natural Resources since 1960, although the Beef River has been stocked since 1937 (Table 1). Three factors available from their records were considered in determining an overall stocking rank for each stream section: 1) years of stocking, 2) total catchable trout per kilometer, 3) total trout per kilometer. Each factor was considered to have equal weight. Trout species was not considered wnen ranklng was aetermlnea. values or one to seven were asslgneo to stream se~tions, one indicating the section with the least amount of stocking intensity and seven showing the most stocking intensity.

20 Table 1. Stocking data of stream sections in the Beef River according to years of stocking, type of trout, and stocking rank. St ream Trout Yrs. of Catchable Brook 1 Total Brook 2 Catchable Brook Trout Total Brook Trout Section 14ater (km) Stocking Trout Trout per Kilometer Per Kilometer 1NT NJ NJ NJ ST SJ SJ w

21 St ream Sect i on Catchable l Other Trout Stocking data of stream sections in the Beef River according to years of stocking, type of trout, and stocking rank. - lata 1 2 Other lrout Catchable Other Trout per Kil ometer Total Other Trout per Kil ometer Total Catchable Trout per Kil ometer Total Trout per Kilometer Stock i ng 3 Rank 1NT NJ NJ NJ ST SJ SJ Excludes Fingerlings: Other trout include brown trout and rainbow trout 2. Includes Fingerlings 3. Stocking rank deternined by ranking four measures of stocking: years of stocking, catchable brook trout, catchable other trout, and total trout per kilometer. Overall stocking ranks were then determined from the sums of ranks for each stream section. 1 = lowest stocking 7 = highest stocking. ~

22 15 A standard sampling protocol was followed at the sampling station. Trout were anesthetized with MS 222 and tissues were sampled immediately. White epaxial muscle was dissected from an area anterior to the dorsal fin. The liver and both eyes were removed, minced, and homogenized. All tissues were diluted, with a 1:l{wt:vol.) in Tris buffer (O.l m Tris, m EOTA, 5 x 10-5 m NAOP, Selander et ale 1971) and ground in mortar and pestle. The samples were labeled and stored on dry ice until return to the laboratory where they were placed in a freezer at - 20 C. Gel Preparation Techniques for electrophoresis were followed from procedures descr-ibed by May et ale (1979). The gels were prepared using 13.2 grams of hydrolyzed potato starch (Electrostarch Co., Madison, Wis., Lot #394) per 100 ml of the appropriate buffer. The standard volume of buffer used per gel was 500 ml. The buffer was placed in a 2000 ml Pyrex flask and heated to 70 C. Approximately 250 ml of the buffer was used to dilute the starch before it was added to the heated buffer. Without proper dilution, clumping occurred. The starch solution was then added to the buffer while rotating both flasks in a circular motion. This motion aided in preventing starch solidification. The starch solution was then heated until a gel was formed. The mixture was degassed under slight vacuum to remove air bubbles, poured into a rectangular plexiglass gel mold (175 mm x 150 mm x 10 mm) and placed in a refrigerator to facilitate solidification. The gels were not used for electrophoresis until they were totally cooled.

23 Each gel was cut approximately 6 cm from the cathodal end with a sharp spatula. Heavy weight filter paper wicks were dipped into the tissue homogenate and placed vertically in the gel cut. The wicks were spaced 1.5 mm apart~ with a maximum number of 20 wicks placed on each gel. The gel mold was then placed on top of a porcelain dissection tray bordered by two plastic buffer trays which contained the positive and negative electrodes. Disposable cloths (eg. Handiwipes) were used to conduct current from the electrode buffer through the gel. A 90 rna current was used to develop each electropherogram. The Clayton buffer system (Clayton and Tretiak 1972) required 200 volts for 3 hours~ and Ridgeway buffer systems (Ridgeway 1979) required 250 volts for 3.5 hours (Table 2). A coating of polyurethane (eg. Saran-wrap) was placed between the dissection tray and the gel to minimize evaporation. An ice pack was fitted into a porcelain dissecting tray and placed on top of the gel to prevent overheating. At the completion of electrophoresis~ the gel was sliced horizontally five times sequentially placing pairs of 1.2-mm tick plastic strips on the sides of the gel and drawing a thin wire through the gel. The slices were placed in plastic trays and the anodal end was marked by a diagonal cut in the upper left corner of the gel. Ihe staining procedure which followed was described ~ Allendorf et al. 1977~ excluding hexose-6-phosphate which was a method of Stegman and Goldberg (1971) (Table 2A). Stains were mixed immediately prior to staining~ poured into the gel and incubated at 37 C for 15 minutes.

24 17 Tab1e 2 Buffer Systems and their associated potential used i~ electrophoresis Grinder Buffer Selander, et ale MTris.001 MEDTA 5 x 10 M NADP ph adjustment ot 7.0 with HCL ln, approximately 10 ml to make 100 ml. Ridgeway Buffer System Ridgeway, F.W. et al., 1979 Potential: 250 volts for 3.5 hours at approximately 40 rna/gel. Electrode Buffer: 0.06 Mlithium hydroxide, 0.3 Mboric acid, ph 8.1 Gel Buffer: 0.03 Mtris, Mcitric acid, ph 8.5 Gels are made using 99% gel buffer and 1% electrode buffer. Clayton Buffer System Clayton, J.W. and D.N. Tretiak, 1972 Potential: 200 volts for 3.0 hours at approximately 30 rna/gel. Electrode BUffer: 0.04 Mcitric acid, ph "6.1. Buffer adjusted to ph with N-(3-arninopropyl )-morpholine. Gel Buffer: Mcitric acid, ph 6.0 Buffer adjusted to ph with N-(3-aminopropyl)-morpholine.

25 Table 2A. Enzyme, tissue, buffer system, and stain solutions used for electrophoresis 1 Enzyme Tissue Buffer System Staining Solution G1ucose-6-Phosphate Dehydrogenase (G6PD) Liver C1 ayton 50 m1 Tris-HCL L:4 0.2 MTris ph mg G1ucose-6-Phosphate 10 mg MgCL 2 * 5 mg NADP * 10 mg NBT * 5 mg PMS Hexose-6-Phosphate Dehydrogenase (H6PD) Liver C1 ayton 38.5 mg Clayton Buffer 15 mg Ga1actose-6-Phosphate * 5 mg PMS * 27.5 mg NADP * 5 mg MgCL 2 * 20 mg NBT Isocitrate Dehydrogenase (IDH) Liver C1 ayton 50 m1 1:4 (buffer:h 2 0) 0.2 MTris-HCL ph8 200 mg DL-Isocitrite Acid 10 mg MgCL 2 * 10 mg. NADP * 10 mg NBT * 2 mg PMS co

26 Table 2A. (Cont.) Enzyme, tissue, buffer system, and stain solutions used for electrophoresis. 1 Enzyme Tissue Buffer Sy stem Staining Solution Lactate Dehydrogenase (LDH) Eye Ridgeway 50 ml Ridgeway gel buffer 15 ml 0.5 MDL-Lactate * 5 mg NAD * 5 mg PMS * 10 mg NBT To make 0.5 DL-Lactate: Use 18.7 g of DL-Lactic Acid 60% Syrup sodium salt, dil ute to 200 ml Malate Dehydrogenase Li ver Ri dgeway 50 ml Ridgeway gel buffer (MDH) 25 ml 0.5 MDL-Na-malate ph 7.0 (ph adjusted to 7.0 with 2 MNa 2 C0 3 ) * 5 mg NAD * 10 mg NBT * 5 mg PMS Abbreviations NAD Nicotinamide adenine dinucleotide * Add immediately before pouring stain NADP - Nicotinamide adenine dinucleotide phosphate on starch gel NBT Nitro blue tetrasolium PMS Phenazine methosulfate lbuffer Systems described on Table 1. \.0

27 20 After readable banding patterns developed, the stain was poured off and fixative was poured over the gels. Approximately 20 ml of a 5:5:1 methanol :water:glacial acietic acid fixative was used to harden the gels for storage. Before refrigeration, the gels were photographed using TR1-X pan film to provide a permanent record of the banding pattern. examined. Polymorphism was observed in three of the five protein systems, The nomenclature used in this report followed the basic design proposed by Allendorf and Utter (1979) for salmonids. An abbreviation was used to designate each protein. In the case of isozymes which are coded for by more than one gene or multiple loci, hyphenated numerals were used. The numbering of multiple loci refers to cathodal to anodal movement; the gene which specified the isozyme with the least anodal (+) migration was designated one, the next two, and so on. Alleles at a particular locus were designated according to the relative electrophoretic mobility. One of the alleles was arbitrarily designated 100. This was usually the most common allele. This arbitrary unit represents the migration pattern of the isozyme coded for by this allele. Other alleles were designated numerical values relative to the 100 unit. An individual producing a product that migrated half as far on a gel was assigned (50). Thus, an allele of the HGPD (hexose-6-phosphate dehydrogenase) locus coding for the least anodal isozyme which specified an enzyme migrating half as far as the 100 unit was designated HGPD-l (50). A heterozygous individuai possessing both alleles (50 and 100) was designated H6PD-l (50/100). When either locus of a duplicated pair was functioning, a comma was used to separate the loci (e.g. IDH-3,4).

28 21 Electrophoretic data were subjected to Hardy-Weinburg equilibrium analysis in the appropriate situations. Equilibrium frequencies using chi-square analysis were used for duplicate loci. To negate linkage disequilibrium as a mechanism disrupting frequency equilibrium, the duplicate loci were examined as a single locus as was the case with IDH-3,4. Statistical Analysis Statistic analysis of electrophoretic data utilized the Biomedical Computer Program (BMDP) for Cluster Analysis and Spearman Rank Correlation. Chi-square contingency tables were calculated using the Interactive Data Analysis (IDA) statistical package. Both BMDP and IDA packages are in the LACE network of the Computer Center at the University. of Wisconsin-La Crosse.

29 22 RESULTS The results of the electrophoretic investigation of 231 brook trout are shown in Figures 2 through 5. The figures are a schematic representation of observed starch gel banding patterns. In two enzyme systems, fixation of the most common allele was observed. The two enzymes, glucose-6-phosphate dehydrogenase (G6PD) and malate dehydrogenase (MDH) were resolved, but did not show variation in fish taken from the stream sections, sampled (Figure 2). The presence of one invariant band in staining for G6PD suggested that one locus codes for the enzyme in liver samples. MDH for brook trout liver samples showed two invarient bands. In the case of the white epaxial muscle dissected in the field, all samples were discarded due to over-dilution with the grinder buffer. The result of over-dilution was a lack of consistent visualization of the protein in question. Whenever a loss of enzyme activity was sufficiently severe to make interpretation of phenotypes impossible, the results for that sample were discarded. Vari ent Enzymes Lactate dehydrogenase. Analysis of observed banding patterns indicated variation at the LDH-3 locus in brook trout eye homogenate samples. A total of three lactate dehydrogenase phenotypes were observed in eye homogenate (Figure 3). Nomenclature for this tetrameric., enzyme has been reviewed with varying results. May et al (1979)

30 MOH-2. MOH-1. _G6PO-1 a b Figure 2. MDH-l, MDH-2, and M)H-2 and G6PD-l phenotypes b) G6PD-l in brook trout liver samples. a) MDH-l N W ':' ~:,~. lo,~_:

31 24 using rainbow trout summarized the conversion of the old nomenclature to the present system, where LDH-3(lOO) = B LDH-3(72) = B ', and l, l LDH-3(86) = B l ". The Bl-subunit in rainbow trout identified by Wright et al. (1975) is designated B 2 -subunit in brook trout. Converting the old nomenclature to the present accepted allelic nomenclature in the Beef River samples~ LDH-3(72) and (loo) were observed. LDH-3(86) was lacking in all field samples including the St. Croix Hatchery fish. The data for lactate dehydrogenase in the Osceola Hatchery fish was supplied by Krueger (1976). His results also indicated that the (86) or (B ") allele was lacking in the Osceola Hatchery. 2 The results of this study indicated the tetrameric structure of LDH and the presence of two alleles expressing three phenotypes (Figure 3~ Table 3). Isocitrate dehydrogenase. In field studies conflicted observations of the mode of inheritance of NADP-dependent found in the liver of rainbow trout have been reported (Allendorf and Utter 1979; Roper et al. 1973). Progeny data indicated that the IDH variation in the liver of rainbow trout is controlled by two disomit loci which code for four alleles (Reinitz 1977). In brook trout~ two loci appear to,code for the liver form of IDH~ one varying and the other apparently invariant (May et al. 1979). The variation found in the liver samples of brook trout for IDH is attributed to two duplicate loci (Figure 3). Two alleles~ including a null allele~ (an allele that either does not produce a protei n or produces a protei n with a great ly reduced activity)

32 Tabl e 3. LDH-3 phenotypes and allelic frequencies with Hardy-Weinburg equilibrium probability values Stream Phenotypes Allelic frequency Section N 72/72 ~/TOO - 100/100 P of a larger X NT 1, > NJ > NJ 3l > NJ 3~ > SST 1) > SJ 3Z > SJ 3~ > St. eroi x 5~ a 3 51 > Osceola > I"\) (J'1

33 be: t =+ -Itt ffhr- rz- ~5J.'!~F'F"'5-~-' '--_- '_'~",,=~- -" -_~.,'",,._'_' LDH-3(100) ~ ~, ~ LDH-3(72) ~ Figure 3. ~ a LDH-3 phenotypes in brook trout eye homogenate samples. b) LDH-3 (100/72). LDH-3 (72/72). o a) LDH-3 (100/100). c N O't

34 27. at the IDH-4 locus were found in all stream sections with the exception of the two Trempealeau County sections (Table 4). A null allele was seen at the other locus IDH-3 in all stream sections with the exception of 2 NJ and 3NJ (Table 5). The interpretation of the phenotypes involving the null allele was based on the intensity of bands. A phenotype involving the null allele in the homozygous state (IDH-4(0/0) ) was not visible during staining. A phenotype involving the heterozygous situation for the null allele (IDH-4(0/100)) showed a reduced staining activity in comparison to the homozygote, IDH-4(100/100). Brandes (1975) proposed that the single-banded phenotype shown in Figure 3(f) was only observed in the Wisconsin population. The author stated that two loci code for gene products with identical migration rates. This report indicates that the null allele at the IDH-4 locus would explain the situation that occurred in Brandes' research. r"~ J Hexose-6-phosphate dehydrogenase. Results of the genetic control of H6PD in brook trout liver tissue indicated one locus coding for this enzyme. The single gene model was assumed for this enzyme. Homozygous individuals each displayed a single band, with electrophoretic mobility dependent upon the allele. In Figure 5, pattern (a) was interpreted as a homozygous individual for H6PD-l(50j50). Th@ handing pattern in (b) is characteristic of an individual heterozygous for H6PD-l(50/100). The pattern seen in (c) was a homozygote individual for H6PD-l(100/100). Research by Shipp (1978) revealed one locus with four alleles, (50), (100), (150) and (200). Shipp's research

35 Table 4. IDH-3 phenotypes and allelic frequencies with Hardy-Weinburg probability values Stream Phenotypes Allelic frequency Section N ~/~ ~/ /100 P of a larger X 2 ~ 100 1NT > NJ 8 0 a 8 >.99 a NJ 29 0 a 29 >.99 a NJ > SST 16 4 a 12 < as SJ 25 6 a 19 < SJ < St. eroi x a 44 < ~O N co _c~~""""_iii<3l!i;;;;;

36 Table 5. IDH-4 phenotypes and alleleic frequencies with Hardy-Weinburg equilibrium probability values Stream Phenotypes P of a 2 Allelic frequency Section N ~/~ ~/loti ~/ / / /200 larger X ~ NT 15 a a a >.99 a NJ 8 a a 2 a 1 5 > NJ a 2 20 <' NJ 41 9 a 1 a 2 29 > ST 16 a a a >.85 a SJ 25 a 2 a > SJ a < St. erai x 54 6 a a 10 < N 1.0

37 -- IDH-4(200) ~ ~ ~ IDH-3(100)~ ~---'~~ IOH-4( 1DO) ~~ ~ a be d e f 9 Fi gure 4. IOH phenotypes in brook trout liver samples. IOH-3 fixed in a-e. a} IOH-4(200/200}. b} IOH-4(~/200} c} IOH-4(lOO/200} d} IOH-4(0/l00} e} IOH-4(0/0} f} IOH-3(~/~} 4(lOO/lOO} g} IOH-3(~/0} 4(200/200} w o

38 Table 6. H6PD phenotypes and allelic frequencies with Hardy-Weinburg equilibrium probability values St ream Phenotypes Allelic frequency Section N 50/50 50/ /100 P of a larger X lnt a > NJ a a > a 3NJ a 4 < NJ a 1 < SST a 1 < SJ a 8 < SJ a a > a St. eroi x > w...

39 "T1 to J:, c Q) (I) "tj c..n r::t ::c _en "U 0 ::c 0'1 "'0 "U =r 0(1) I ~... 0 M -'< c..n "'0 _orov' O~, -r::t 0.. 0,... ',' ('l.. M -, t, 0 :l:, c ::c M ~1" 0'1 "U , I <... (I) ~ ;,~ ", -... V' o III 03 _"'0... 0(1) OV' -" III - ::c 0'1 "U 0,... -c..n 0 c..n 0- ('l III c;r? -4-C1I 0- ~ "tj o o-

40 33 showed that only H6PD-1(50 and 100) were present in a Lawrence Creek, Wisconsin population. Variation at H6PD in the present study resulted in three phenotypes at vary i ng frequency (Table 6). Hardy-Weinburg Equilibrium The basic assumption of the Hardy-Weinburg law is that diploid genotypes observed in a population are determined solely by random association of alleles. The determination of the expected genotypes is derived from the allele frequencies. Given the genetic hypothesis as stated above, allelic frequencies were calculated for each of the samples, and Chi 2 tests were performed to test for random union among gametes according to the Hardy-Weinburg law. A description of the errors involved with this model js given by Fairbain et ala (1980). Not all autosomal loci considered separately attain Hardy-Weinburg equilibrium in genotypic frequency after one generation of random mating. If two alleles are involved, as with LDH-3 (72) and (100), and if they occur in the population with frequencies p and q, then the genotypes (72/72), (72/100), and (100/100) should occur in the population with frequencies p2, 2pq, and q2, respectively. A good t; "' ~ ~,J' '1'1 '~, J: b fit with Hardy-Weinburg expectations was shown for all stream sections at the LDH-3 locus (p >.05) Table 3. The assumption of random mating with two loci was analyzed at IDH-3,4. The Hardy-Weinburg equilibrium frequencies at each locus were considered jointly. The results rejected the genetic hypothesis,

41 34 thus linkage disequilibrium was suspected. To eliminate the effects of linkage disequilibriijm. the duplicate loci at IDH-3,4 were considered as individual loci (Tables 4,5). At the IDH-3 locus, adherence to equilibrium was obtained in trout ~rom stations 2NJ, 3NJ, and 4NJ (p >.05). Lack of fit in Hardy-Weinburg equilibrium in stream sections lnj, 5ST, 6ST, and 7SJ was due to an excess of the observed homozygotes IDH-3 (~/0) (p <.05). The St. Croix Hatchery fish also did not conform to the Hardy-Weinburg equilibrium showing an excess in phenotypes (0/~) and (0/100) (p <.005). While most populations coincided with Hardy- Weinburg expectations at the IDH-4 locus, samples from stations 3NT, 4NT, and 7ST did not. Lack of fit was due to the presence of expected values lower than observed phenotypes. The St. Croix Hatchery fish also failed to conform to Hardy-Weinburg equilibrium at IDH-4 due to a lack of (100/100) phenotypes (p <.005). At the H6PD locus, stream sections lnt, 2NT, and 7ST were similar to Hardy-Weinburg expectations (p >.05) (Table 6). The St. Croix Hatchery fish also conformed to the expectation (p >.05). Lack of fit in sections 3NJ, 4NJ, 5ST, and 6SJ was due to expected values less than one for the rare phenotype (100/100) or to the excess of phenotype (50/100) (p (.01). Genetic Comparison of Hatchery and Field Collections Chi 2 contingency tables using the phenotypic frequencies at LDH-3,IDH-4, and H6PD were used to compare the genetics between the

42 35 T.ble 7. x 2 test of homog~neity with the St. Croix H.tchery using IOH-4 phenotypic frequencies. Stream Sect ion X 2 d. f. P of l.rger X 2 lnt <.005 2HJ <.005 3NJ <.005 4NJ <.005 SST <.005 6SJ <.005 7SJ <.005 Table 8. x 2 test of homogeneity-with the Osceola population using ldh-3 phenotypic frequencies. St ream Secti on X 2 d. f. P of larger X 2 INT ).25 2HJ ).31 3NJ ).81 4NJ ).05 SST ).05 6SJ <.005 7SJ <.005 ".~., St. Croix <.005,ii.' '~ ~.1, hatchery brook trout and the native brook trout. The test of genetic homogeneity was performed with the St. Croix Hatchery fish using all three loci. Data for the Osceola Hatchery fish was available only for- LDH-3. The p val ue expressed the probabll ity that the Chi 2 would be larger in another random sample, if the frequencies were the same between the groups. Testing between field samples and St. Croix Hatchery fish at the IDH-4 locus revealed statistically significant heterogeneity in all stream sections (Table 7). The hatchery population had a

43 36 Table 9. X 2 test of homogeneity with the St. Croix Hatchery using LDH-3 phenotypic frequencies. St ream Sect i on x 2 d. f. P of a larger X 2 1NT <.005 2NJ <.005 3NJ <.005 4NJ <.005 SST <.005 6SJ <.005 7SJ <.005 higher frequency in the IDH-4 (200) locus (Table 5). IDH-4 (200) predominated in all sections with the exception of SST. At the LDH-3 locus, both alleles were present in both the St. Croix and Osceola Hatchery fish (Table 3). In the Osceola and the St. Croix fish, LDH-3 (100) was the most common. A variable allelic frequency was seen (Table 7). Statistically significant differences existed between the phenotypic frequencies of the St. Croix Hatchery and all of the stream sections in the Beef River (Table 8) (p <.005). Comparisons between the LDH-3 locus of the Osceola Hatchery and the stream sections indicated lnt, 2NJ, and 3NJ to be the most statistically similar sections (Table 9). Also, significant heterogeniety existed between the two hatchery populations (p <.005).

44 37 2 Table 10. X test of homogeneity with the St. Croix Hatchery using H6PD-l phenotypi c frequenc i es. 2 2 Stream Section x d. f. P of a larger X 1NT >.32 2NJ > 18 3NJ >.01 4NJ >.005 5ST >.12 6SJ <.005 7SJ >.005 H6PD-l(50) was the most common of the two alleles present in both the field collections and the St. Croix Hatchery fish (Table 6). Trout from sect ions 2NJ and 7SJ were fi xed for H6PD-l. Phenoty pi c comparisons showed fish from sections lnt, 2NJ, and 4NJ to be the most similar to the St. Croix Hatchery fish (Table 10). Within-stream variation. Three equidistant samples from a 3.2 km stream section were taken to identify breeding structure within the stream. Both IDH-4 and LDH-3 loci were used to analyze for withinstream variation. Differences did exist within a relatively short distance in the stream (Table 11). Statistically significant (p <.05) heterogeneity exists at both loci when the three stream sections were considered jointly. The most homogeneous unit at both

45 Table X homogeniety testing at the LDH-3 and IDH-4 locus within stream sections Stream Section X 2 LDH-3 df p X 2 IDH-4 df 2NJ 3NJ 4NJ I >.01 I >.01 2NJ 3NJ > ).50 3NJ 4NJ > <.05 2NJ 4NJ <.005 I <.05 2NJ 3+4NJ 2+3NJ <.05 I ). 14 4NJ <.05 I >.01 p loci was 2NJ and 3NJ. At the IDH-4 locus, the upstream section (4NJ) had a higher frequency of the null (~) allele than the downstream collections (Fig. 6). An inverse relationship exists at the LDH 3 locus (Fig. 7). Fish sampled from section 2NJ to 4NJ had an increased frequency of the LDH-3(lOO) allele and a decreased frequency of the LDH-3(72) allele. Between-stream variation. The LDH-3 lo~us indicated similar allelic frequencies in 13 of the 21 stream sections examined (Table 12). The allelic frequency of LOH-3(100) increased while the (72)

46 o 1 km r""iw""\iii ~ Within Stream Variation o IDH n 200 2NJ 4NJ ~ 'r.~..f., 1 -, m~: 2NJ 3NJ 4NJ Figur.e 6. Percent frequency of IDH-4 variation within three equidistant stream sections in the North Fork of the Beef River W 1.0 ~ ~~~"... ",

47 -,'-." ~.,';7v-i i"fr:a ItS L ii [,_jk.hi!i5;!agqfii!iietiiiji;bi\i".(?::;i~l Within Stream Variation LDH o 1 km ~ 4NJ ~:I.... fr.q NJ 3NJ 4NJ Figure 7. Percent frequency of LDH-3 variation within three equidistant stream sections in the. North Fork of the Beef River.j:::a c

48 :s:;;,: h ~J #4 XH;f7"A Fil"", Ol, ;;"il ","",w,,,?,, "7?',;;r " " -~-.,..,.'-'!IIl"!ff!Il;~'fM-I~'~'::"lfl!!!lfY!''if"4iif;:7:'frj;: :~:i::;:r::f'irl?%':._ '. >y,~ :3"''' '' "'1 Between Stream VarIation,. Ir North FDrk lnt 2NJ 3NJ.NJ LDH-5 INJ ~NJ.N"" 12m 100 ;~II.R,. Ir.... Sou I" F o,r k A.1 SST esj Figure 8. Percent frequency of LDH-3 variation between North and Sout~ of the Beef River Forks.l'>o...

49 42 allelic frequency decreased (Fig. 8). When analyzing the South Fork and moving from SST and 7SJ, LDH-3(lOO) decreased while LDH-3(72) increased. No distinct relationship was seen between the geographical position and allelic frequency distribution at LDH-3. At the IDH-4 locus, only 5 of 21 comparisons were homogeneous (Table 13). Similarity between sections was evident with the sections in close geographical position to one another forming homogeneous groups. Two groups, lnt-5st and lnt-6sj, did show homogeneity even though the sections are located on different forks (Table 13). lnt and SST were the only stream sections not possessing the null allele at IDH-4 in the Beef River (Fig. 9). The frequency of the null allele increased toward the upper headwater stream sections. The Lake Martha Dam forms a physical barrier between the North '~'i;,;. 'Il ;1" and South Forks. Movement of fish upstream beneath the dam on the I~I: South Fork is not possible, but movement downstream over the dam with the subsequent movement of fish into the North Fork is conceivable. Genetic Distances Between Stream Sections Cluster analysis was performed on the allelic frequency based on a measure of similarity between the frequencies. The clusters were formed according to the amalgamation distance or the distance ~::;I~.,~ b~tween allelic frequencies for each stream section. Each ~trp~m section was considered a case. The distance between two cases or clusters was defined as the Chi-square test of equality of the two sets of frequencies.