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Genetic and environmental mechanisms controlling the lakeward migration of young rainbow trout (Salmo… Kelso, Bryan William 1972

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THE GENETIC AND ENVIRONMENTAL MECHANISMS CONTROLLING THE LAKEWARD MIGRATION OF YOUNG RAINBOW TROUT (Salmo gairdneri) FROM OUTLET AND INLET REARING STREAMS by BRYAN WILLIAM KELSO B.Sc. University of British Columbia, 1970 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Zoology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA 1972 In p r e s e n t i n g t h i s t hes i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y sha l l make i t f r e e l y a v a i l a b l e f o r reference and s tudy. I f u r t h e r agree t h a t permiss ion f o r ex tens ive copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be al lowed w i thou t my w r i t t e n pe rm iss ion . Department o f The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8 , Canada ABSTRACT The upstream-downstream response to water current exhibited by rainbow trout fry in inlet and outlet streams of Loon and Pennask Lake stream systems was studied in experimental laboratory performance channels. Analysis of d i a l l e l tables, developed by crossing seven different inlet and outlet spawn-ing stocks from the two stream systems, demonstrates additive genetic d i f f e r -ences between the two stocks with respect to current response. Tests performed i n daylight showed a net upstream movement for a l l stocks, but far greater for outlet compared to inlet fry. At night, inlet fry showed a very strong downstream movement while outlet fry showed very l i t t l e movement, similar to their behavior i n the f i e l d . Further analysis of the d i a l l e l table, when a l l the stocks were tested at three temperatures (low: 5C, medium: IOC, high: 17.5C), showed that temperature both in daylight and darkness tended only to change the degree of upstream or downstream movement of the f i s h , rather than the direction of movement. In daylight, upstream movement for a l l stocks was greatest at low temperature and least at high temperature. In darkness the greatest downstream response was at high temperature. However, at high temperature outlet fry moved farthest upstream in daylight while in darkness inlet fry moved farthest downstream. Other possible controlling mechanisms (sudden temperature rises in the outlet creek, water source, abundance of food, genetic differences in l i v e r lactate dehydrogenase) are considered. The d i a l l e l analysis suggests that there are genetic differences in the current response between the inlet and outlet stocks and that water tempera-ture plays only a minor role in the migration of rainbow trout fry to the lake. i i i TABLE OF CONTENTS Page TITLE PAGE i ABSTRACT i i TABLE OF CONTENTS i i i LIST OF TABLES v LIST OF FIGURES v i ACKNOWLEDGEMENTS ' v i i i I INTRODUCTION 1 II MATERIALS AND METHODS 4 A. Collecting and Holding Adults 4 B. Crossing Procedure 6 a) D i a l l e l cross 6 b) Within population crosses 9 C. Performance Channel 11 D. Method for Behavioral Tests 13 III GENETICAL ANALYSES AND STATISTICAL METHODS 14 IV SCORING SYSTEM 17 V RESULTS 18 A. Behavioral Responses of Fry i n the Experimental Channels 18 B. Survival 19 C. Performance Tests 19 a) Pure stocks 19 b) D i a l l e l crosses 26 i) Hayman's analysis of the d i a l l e l tables 26 i i ) The factorial analysis of variance 34 VI DISCUSSION 43 A. Genetic Effects 44 B. Maternal Effects 50 C. Temperature Effects 50 D. Other Possible.Effects 52 a) Water sources 52 b) Other environmental effects 53 i v Page VII CONCLUSIONS 53 BIBLIOGRAPHY . 55 APPENDICES 57 V LIST OF TABLES Table I The mean and range in fork length, (mm) for parents of the 1971 d i a l l e l cross. Table II The mean and range in fork length (mm) for parents of the 1971 self-crosses. Table III Analysis of variance on the net scores of the behavioral tests on the pure stocks using the three factors where A = stocks, B = light and C = temperature. Table IV The expected mean squares and the variance components for the three-factor analysis of variance on the pure stocks, n = 12, a = 6, b = 2, c = 2. 2 Table V Model for expectations of mean squares of a n d i a l l e l cross, when the parental lines are fixed, for Hayman's analysis of variance. Table VI Levels of significance for Hayman's analysis of variance of the d i a l l e l crosses for the net scores. "P" means the item was tested against the pooled interaction mean square. Table VII Percent of the variance components for Hayman's analysis of the d i a l l e l tables of the net scores. Pooled mean square used as error variance. Table VIII Three-factor analysis of variance table on the net scores where A = males, B = females, C = temperature, for the daylight and darkness runs for both Cross I and Cross II. Expected mean squares for three-factor analysis of variance, fixed model, and the variance components for the four analysis of Cross I and II for daylight and darkness, n = 2, a = 7, b = 7, c = 3. The percent of the adults used i n the d i a l l e l cross which contain three types of l i v e r lactate dehydrogenase (L.D.H.). See text for explanation of the three types of L.D.H. Table IX Table X v i LIST OF FIGURES Fig. 1 Loon Lake and Pennask Lake stream systems showing major rainbow trout spawning areas (>?^fi. ) . Fig. 2 The arrangement of the 1971 d i a l l e l cross. The leading diagonal (X) represents the self-crosses while the rest are interpopulation crosses. Fig. 3 The standard 25-compartment performance channel (entrance and exit of each compartment staggered). The c i r c l e in the upper plan view shows the compartment used to release fry. Fig. 4 Net scores for the behavioural tests for the current response of the pure stocks. Solid circles and bars represent means and ranges, respectively of 12 replicas. Fig. 5 F i r s t order interactions of the analysis of variance (Table III) on the pure stocks; significance indicated by the degree of crossing between lines (see Sokal and Rohlf, 1969 p.355). Fig. 6 Upstream-downstream preference tests on three stocks of Loon Lake fry conducted in "Brannon-type" (Brannon, 1967) performance troughs during the summer of 1969. Water temperatures = 9.4C, velocity = 6.7 cm/sec, water source =-.-Loon Creek Hatchery spring water. Fig. 7 The mean scores of both crosses for the "leading diagonal" (see Fig.2) and some of the hybrids for the daylight and darkness tests conducted at the high temperature (17.5 ± 1.5C). Single symbols represent the self-crosses, paired symbols represent the hybrids. Fig. 8 The mean scores of both crosses for the "leading diagonal" and some of the hybrids for the daylight and darkness tests conducted at the medium temperature (9.7 ± .5C). Single symbols represent the self-crosses, paired symbols represent the hybrids. Fig. 9 F i r s t order interactions of male (A) by female (B) in the factorial analysis of males by females by temperature. Fig. 10 Firs t order interactions of male (A) by temperature (C) in the factorial analyses of males by females by temper-:, ature. Fig. 11 Fir s t order interactions of female (B) by temperature (C) in the factorial analyses of males by females by temperature. v i i Fig. 12 The mean scores of both crosses for the "leading diagonal" in a linear regression of high temperature and low tempera-ture for daylight and for darkness. Question marks means i t i s uncertain i f the stock should be called an upstream or a downstream stock. Fig. 13 Net score of the current response of Loon Inlet fry which contain two separate phenotypic forms of l i v e r lactate dehydro-genase. Tests conducted in 25-compartment performance channels. Circle and bar represent mean and ranges, respectively of 12 replicas. Data provided by T.G. Northcote; to be published elsewhere. v i i i ACKNOWLEDGMENTS The Fish and Wildlife Branch of Bri t i s h Columbia and the National Research Council of Canada provided financial support for this study. Dr. J.R. Calaprice of the Fisheries Research Board at Nanaimo, B.C. granted the use of their f a c i l i t i e s at their genetics hatchery at Rosewall Creek on Vancouver Island. Also, Mr. R.A.H. Sparrow, Fish and Wildlife Branch biologist in charge of Fish Culture, granted permission to u t i l i z e Look Creek Hatchery f a c i l i t i e s . This cooperation was greatly appreciated. Numerous associates, including G.R. Peterson, R. Norman, R. Land, D. Smith, G. Card, M. Flynn and G. Ennis aided in the collection of the adult fi s h and i n the performing of the d i a l l e l cross, and their assis-tance was greatly appreciated. Much assistance was also given during the laboratory tests by A. Solmie of the Fisheries Research Board. A great deal of aid was given in the computer programming of the st a t i s t i c a l analysis by Mrs. D. Lauriente of the U.B.C. Computer Centre and Messrs. J. Thompson, F. Nash and Mrs. A. Sandres of the Computer Centre at the Fisheries Research Board in Nanaimo. Dr. C F . Wehrhahn read the manuscript and made valuable suggestions during the course of the study. The support, advice and patience of Dr. J.R. Calaprice has been greatly appreciated throughout a l l phases of this study. I wish to express my gratitude to my supervisor, Dr. T.G. Northcote, for his guidance and constructive criticisms throughout a l l phases of the i x study and the preparation of the manuscript. Finally, I wish to express my gratefulness to my wife, Verna, for her understanding, patience and assistance throughout a l l phases of my studies. I INTRODUCTION Migration of animals from one region to another for breeding or feeding has been the object of study of many zoologists and naturalists for some time. One group of migrating animals are the many species of freshwater and marine fish that migrate to spawn during their l i f e cycle. Some of these fishes, such as eels, herring, cod, plaice and tuna, travel hundreds of miles (Harden-Jones 1968). Of most interest on the North American Pacific Coast are the anadromous Salmonidae and the mechanisms that control their migrations. Hoar (1953) states that chum, pink, and sockeye fry migrations down-stream are a passive movement. He believes that this is brought about when they lose visual contact with the bottom due to low light intensity at night. However, the adult sockeye not only migrate "up" inlet streams to spawn, but also move "down" outlet streams to spawn. Thus, the young, must either migrate "downstream" or "upstream" from their incubation stream to the rearing lake. The occurrence of inlet and outlet spawning is not only associated with sockeye salmon, but also with brown trout, Salmo trutta; grayling, Thymallus articus; white and longnose suckers, Catostomus commersoni and C. catostomus; as well as rainbow trout, Salmo gairdneri (Northcote, 1962). Because the fry of the outlet spawning adults must move upstream, their movement cannot be passive but must be a deliberate, controlled move-ment. Therefore, a fundamental problem in migratory behaviour was to determine the mechanism that controls these marked behavioural differences between fry of the same species. Northcote (1962) made two postulates as to the control of lakeward 2 migration, (1) genetically distinct outlet and inlet stocks of which each had an appropriate innate behavioural response which resulted in the movement of young into the lake, (2) genetically similar outlet and inlet stocks responding to environmental differences between streams which pro-duced the appropriate current responses and other behaviour characteristics of the young as they move into the lake. Raleigh (1967) added a third postulate which stated that there were gentically distinct outlet and inlet demes maintaining innate behavioural responses which may be modified by the environment. In Northcote's original study (1962) on the inlet and outlet progeny of rainbow trout, he concluded that the mechanisms controlling migration were associated with environmental differences between streams rather than genetic differences between spawning stocks. The main environmental factors were temperature and photoperiod. Cool water temperature, and long day lengths apparently induced downstream movement while short day lengths and warm water temperatures were associated with upstream movement of fry. However, he noted in his 1962 paper and again in his later studies (1969), that different migratory traits apparently have developed within a few years from "single stocks" of trout. He stated that in another inlet-outlet stream system, Pothole Lake, there could be a selectivity for a genetically controlled upstream-downstream migration. Furthermore, in 1969 he found that there were marked differences in migratory behaviour between "above f a l l s " and "below f a l l s " populations which were not controlled by obvious environmental factors. From this, he concluded that there might be gene-t i c a l l y controlled, as well as, environmentally-induced behavioural mechanisms operating in the Loon Lake system. Brannon (1967), in a study of upstream and downstream movement of sockeye fry, concluded that there was a deliberate, genetically controlled 3 movement from incubation area to the nursery lake. Also, Raleigh (1967, 1971) and Raleigh and Chapman (1971) concluded from their studies of sockeye salmon, cutthroat trout, and rainbow trout that the migratory behaviour of salmonid fry from natal to rearing areas is under innate control subject to modifications by the environment. In 1969 a preliminary study suggested and directed by T.G. Northcote (Kelso, MS, 1970), showed that there was some genetic control in the migra-tory behaviour of stocks of rainbow trout of Loon Lake, B.C. Recently, Calaprice (1972a and 1972b) designed an experiment using biometrical gene-tics to describe differences in heritable factors that occur among popula-tions of sockeye salmon. He found that there were additive genetic differ-ences and maternal effects that influenced the survival of the young (1972a) as well as heritable differences in current response among the progeny from adults collected in different streams (1972b). Previous studies on rainbow trout of the Loon Lake system had shown both environmental and genetic controls in migration of the fry. Also Raleigh's (1967) and Brannon's (1967) work on young sockeye salmon migration showed a genotype-environmental interaction. The diallel analysis used by Calaprice (1972b) partitions the total variation between populations into genetic and environmental components; the genetic component consisting of additive and dominant effects, while the environmental component is maternal (or paternal) effects and those effects brought about by the surrounding environment. This method was used to test Northcote's (1962) hypothesis for genetic differences in current response between stocks. If there are no significant differences in response between families, then any varia-tion that exists must be environmental and thus tests his second hypothesis. The analysis should also show i f there are significant interactions between 4 the genetic and environmental components, testing Raleigh.'s (.1967) hypothesis. To increase the number of populations, a second lake, Pennask Lake, was also used in the study. In a l l , five spawning populations from Loon Lake plus two spawning populations from Pennask Lake were used to set up a 7 x 7 d i a l l e l cross to test for genetic and environmental mechanisms that might control the current response of young rainbow trout fry. II MATERIALS AND METHODS A. Collecting and Holding Adults Nearly mature spawning rainbow trout were collected from five different spawning areas on the Loon Lake stream system (Fig. 1), during May and early June, 1971. These areas were Cl) Loon Inlet Creek, (2) Thunder Creek, (3) Loon Outlet (Outlet Trap 1), (4) Hihium Creek (from 50 to 100 meters upstream from the confluence of Hihium and Loon Outlet Creek), and (5) 50 to 200 meters downstream from the confluence of Hihium and Loon Outlet Creeks. Also, an inlet and an outlet stock * was obtained from Pennask Inlet at the B.C. Fish and wildlife Branch hatchery trap situated approximately 100 meters upstream from the lake and from Spahomin Creek at a trap situated at the mouth of the Creek (Fig. 1). The adults were held in eight fiberglass tanks (approximately 1.2m x .6m x .45m), and i n two wooden ponds (approximately 5m x 1.2m x lm) at the B.C. Fish and Wildlife Branch Hatchery about 5 miles southwest * For purposes of this study each group of fi s h from these areas has been called a separate "stock". 5 6 of Loon Lake. A minimum of twenty pairs of adults were collected from each stock. B. Crossing Procedure a) D i a l l e l cross The d i a l l e l cross consists of making a l l possible matings between n pairs of adults. In 1971 two such crosses were performed separately and the eggs and sperm of each stock were pooled. For each cross, five adult pairs were used and the eggs were divided into two replicas. In some cases i t was necessary to use more than five males to obtain enough sperm and i n other cases fewer than five females were mature enough to provide ripe eggs. The number of adults used and their mean lengths are shown in Table I. Crosses between the seven areas were performed as il l u s t r a t e d in Fig. 2. Each stock of eggs were placed i n separate 2 l i t e r p lastic freezer cartons and were gently stirred so that the pooled eggs were as equally distributed as possible. The pooled sperm of each stock was placed in separate styrofoam cups and also stirred. Two trays 1.2m x 1.2m were prepared so that each tray held forty-nine plastic cartons. The rows and columns of the trays were colour-coded with the rows designated for males and the columns designated for the females. The pooled eggs of each stock were f i r s t divided into two replicas. Each replica of eggs in turn was then divided into seven approximately equal lots and placed in their appropriate cartons in the tray Ccolumns Fig. 2). After a l l seven stocks of eggs were placed in the tray, they were then f e r t i l i z e d with the aid of seven different plastic disposable hypodermic syringes, one for each stock (rows, Fig. 2). The tray was then placed under the egg washer (water temperature IOC) and the eggs 7 Table I. The mean and range i n fork length (mm) f o r parents of the 1971 d i a l l e l cross. Stock Cross I Female Male no. mean range no. mean range Pennask I n l e t 6 313 280 — 342 8 322 236 — 350 Loon I n l e t 6 338 317 - 364 6 347 263 - 404 Thunder Creek 6 344 330 - 365 6 339 320 - 360 Pennask Outlet 5 288 260 - 306 6 301 295 - 313 Loon Outlet 4 310 285 - 331 6 285 201 - 337 Below Hihium 3 288 280 - 295 7 231 192 - 300 Hihium Creek 6 291 260 - 320 6 229 165 - 360 Cross II Pennask I n l e t 6 306 287 - 340 6 285 261 - 297 Loon I n l e t 5 361 338 - 405 4 250 195 - 351 Thunder Creek 6 307 277 - 343 6 280 240 - 300 Pennask Outlet 5 307 282 - 383 6 306 258 - 330 Loon Outlet 5 310 290 - 332 6 286 232 - 379 Below Hihium 4 278 210 - 342 15 235 191 - 284 Hihium Creek 7 316 260 355 15 235 168 — 360 8 Fig. 2 The arrangement of the 1971 d i a l l e l cross. The leading diagonal (X) represents the self-crosses while the rest are interpopulation crosses. FEMALE Pennask Inlet Loon Inlet Thunder Inlet Pennask Outlet Loon Outlet Below Hihium Hihium Pennask I n l e t X Loon I n l e t X Thunder I n l e t X Pennask Ou t l e t X Loon Outle t X Below Hihium X Hihium X 9 were water hardened. The next replica was then begun. After approxim-ately ten minutes of washing, the egg cartons were capped and placed in styrofoam boxes and packed in ice. The total number of egg cartons was 196 (2 ( 7 x 7 x 2 ) ). Egg taking and crossing took approximately four hours. The eggs were then transported by truck 525 km to the Fisheries Research Board's genetic hatchery at Rosewall Creek on Vancouver Island. Transportation time was approximately ten hours. The eggs were placed at random in 196 separate rearing tanks. One l i t e r of water per minute circulated through each tank. The eggs were treated twice weekly with malachite green unt i l just before hatching. Up to swim up, ie. the time when fry emerge from the gravel, water temperature ranged from 6.4° to 11.8°C with a mean of 9.2°C. After swim up ( 476 CTU^) the incubation baskets were removed and outside standpipes were installed. Fry were fed frozen commercial hatchery mash supplemented with frozen brine shrimp. b) Within population crosses As well as the d i a l l e l cross, males and females from each stock were mated and for purposes of this study, are called the "pure stocks". The eggs for each stock were pooled and f e r t i l i z e d with the pooled sperm of the same stock. The number and lengths of the adults are given in Table II. The eggs were held at the Loon Creek Hatchery (water tem-perature 10°C) unt i l they were eyed or, in some cases, had already reached the alevin stage and were then transported to the Rosewall Creek hatchery. Degrees centigrade temperature units (sum of the degrees centigrade per day the water was in the rearing tanks above zero degrees centigrade). 10 Table II. The mean and range in fork length (jam) for parents of the 1971 self-crosses. Stock Female Male No. Mean range No. Mean range Pennask Inlet 5 316 299 - 349 5 305 278 - 320 Loon Inlet 4 334 302 - 353 5 348 315 - 367 Thunder Creek 5 304 283 - 324 3 351 334 - 366 Pennask Outlet 2 293 280 - 306 2 321 310 - 332 Loon Outlet 4 289 284 - 292 4 294 228 - 343 Below Hihium 6 294 274 - 308 4 294 222 - 331 Hihium Creek 2 314 300 - 328 2 255 205 - 305 11 Performance Channel The experimental apparatus used (Fig. 3) was designed by J.R. Calaprice (1972b). It consisted of a wooden trough, 15 cm wide by 3.05 m long and 15 cm high; divided into twenty-five compartments. A 2.5 cm hole lead from one compartment to the next in a staggered position. This prevented a direct flow of water from one end of the trough to the other end, as the water would "swirl" around in a com-partment before running into the next one. Thus, a fish had to "seek out" the entrance into the next opposing compartment whether i t was moving upstream or downstream. Twelve such performance channels were used in two banks of six. A dark room, approximately 3.7mx3.7mx2.7m was constructed by covering a wooden framed area with 4 mil black polyethylene plastic. This was divided into two rooms by a polyethylene partition, each hav-ing a separate entrance and containing a bank of six performance troughs. Al l twelve channels were fed by the same headtank which protruded through the centre partition into each room. Controlled flow (1250 ml/min) was maintained to each channel giving a velocity of 7.4 ± .5 cm/sec. through the compartment openings. The lighting used for each bank of performance troughs consisted of five 150 watt, 125 volt, projector flood lamps. These were hung on the centre partition wall and directed upward onto a white glossy c e i l -ing to give indirect lighting. This eliminated a l l shadows in the trough, but not necessarily a l l light gradient. The lights were con-trolled from outside the room with an automatic timer or a powerstat (Superior Electric Co. Type 110). The timer took one-half hour to increase to maximum or decrease to minimum light intensity. 12 Fig. 3 The standard 25-compartment performance channel (entrance and exit of each compartment staggered). The circ l e in the upper plan view shows the compart-ment used to release fry. Outlet trap Inlet Outlet release point Inlet trap 13 The tests were conducted at three different temperature regimes 3.5-6.5 C, 9.0-10.5 C, and 16.0-19.0 C. The high temperature was obtained by recycling 10 C Rosewall Creeks water and heating i t with a 1000 watt emersion heater (Waage Electric Inc. Model SF100). The medium temperature was maintained by combining river and ground water, while the low temperature was maintained by recycling ground water cooled by a 'Blissfield refrigerant unit Model BHL-909-B. Method for Behavioral Tests Testing began shortly after "swim up" at the high temperature (614 C T.U.) and ended at 739 C T.U. of development. The medium temperature tests were run from 759 C T.U. to 836 C T.U., and lower temperature tests from 870 C T.U. to 968 C T.U. of development. Fry tested ranged between 20 and 30 mm fork length and a l l had started to feed. In each test, a maximum of 20 fry were used, depending upon the number of survivors per tank. Fry were placed in the central com-partment of the test channel and held there by placing aluminum strips over the exit holes. A test was f i r s t performed under daylight conditions. Then, the same f i s h were placed back into the troughs and tests run again i n darkness. For the daylight tests the experiments were begun in complete darkness and the lights were slowly increased to maximum intensity over a one-half hour period. Fifteen minutes after the test was begun (when the light was at half i t s maximum inten-sity) the screens on the exit holes were removed and the f i s h were free to move upstream or downstream. After 2% hours from the start, the 1 * positions of the fish, were noted with, the lights dimmed to 55 volts and after a l l the fish, in the twelve troughs had been counted the fish were removed and placed back in the centre compartment. For the response of the fry in darkness, the lighting was the reverse. Upon completion of this test, the fry were placed back in the rearing tanks. For tests in which the water temperature in the experimental troughs was higher than the water in the rearing tanks, the fry were collected approximately 45-60 minutes in advance and held in plastic freezer cartons until the temperature warmed to within 2C of that in the experimental trough. Flow rates were measured both before and after each test. Three to four sets of experiments were done per day for a total of 36 to 48 individual tests. I l l GENETICAL ANALYSES AND STATISTICAL METHODS There have been several experimental designs devised for estimating the genetic and/or environmental variation in plant and animal populations (Kearsey 1965). The design of this experiment was similar to the North Carolina Design 2, where a l l the mn progeny families that were obtained by crossing m males with n females were raised, ie a ful l dial le l cross. 2 Wearden (1964) defines a ful l dial le l as ".. .p possible matings among a set of parental lines including 1/2 p(p-l) pairs of reciprocal crosses." Two methods of analysing the dial le l cross are the Hayman analysis and the factorial analysis (Wearden, 1964). These are models for maternal and for reciprocal effects. 15 Hayman's analysis of the model for reciprocal effects gives the 2 most powerful test for the genetic contribution (o~) but the f a c t o r i a l analysis for maternal effects gives the best test for maternal factors 2 (cr ) because i t t e l l s whether the variance is due to the maternal or to m the paternal effects of the parent (Wearden, 1964). For Hayman's analysis of variance (Hayman, 1954a) there are six basic assumptions: (1) Diploid segregation; (2) No-difference between reciprocal crosses; (3) Independent action of non-allelic genes, and i n the d i a l l e l cross; (4) No multiple allelism; (5) Homozygous parents; (6) Genes independently distributed between parents. His analysis yields seven s t a t i s t i c s : "A" — genetic variation amongst parents (additive variation), "B" — variation in reciprocal sums not ascribed to A or non-additivity (dominance), "C" — average maternal effects of each parental line, "D" — variation in reciprocal differences not ascribed to C. On the assumption that the genes are independently dis-tributed between parents, the "B" term is divided into three separate s t a t i s t i c s : "b^" — testing the mean deviation of Fl's from their mid-parental values and is significant only i f the dominance deviations are directional, "b^" — testing whether the mean dominance deviation of the F l from the mid-parental values within each array differs over arrays (i.e. gene assymetry or dominance at some of the l o c i ) , and "b^" — testing that part of the dominance deviation unique to each F l . However, because of the assumption underlying these latter three s t a t i s t i c s , they have been omitted from the main discussion of the results. For those who wish to carry the analysis to i t s f u l l extent the b^, b2> b^ terms have been l e f t i n the anova table in Appendix 3. 16 Hayman's analysis calls for the mean squares for each main effect to be tested for significance against its own Interaction over blocks (environ-mental component). However, to increase the degrees of freedom the error variances, where homogeneous, may be pooled to give a block interaction mean square as a common error variance (Hayman 1954a). The computer program used was written by J.R. Calaprice of the Pacific Biological Station, Nanaimo, B.C. Hayman (1954a) gives a graphical analysis of the dial lel cross which can show either the additive or dominant effects by the use of' a variance-covariance graph. However, i t too depends upon the same assumption of independent assortment of genes, so has been omitted from this analysis. A further point is that the experiment is regarded as a fixed effects analysis because the streams containing the adults were specific streams picked from the Loon Lake and from the Pennask Lake areas and was not a sample of streams from a large population. The factorial analysis of the data is based on the model for maternal effects (Wearden 1964), but is modified to include also the environmental effects of temperature. The interaction terms not only show the genetic contribution of the parent stocks but also show how the animals vary in response to a specific environment, or in other words a genotype by environ-mental interaction. The expected mean squares were calculated by the rules set down by Sokal and Rohlf (1969) for a multi-factorial fixed effects analysis of variance. The statistical analysis was carried out at the Pacific Bio-logical Station in Nanaimo, B.C. using the program for factorial anova from Sokal and Rohlf, number C A 3.5. IV SCORING SYSTEM In this analysis, no test for scaling (ie mathematical transformations) was made. However, the analysis was performed on the raw data using several different scoring procedures. These a l l produced similar F-ratios. The f i r s t procedure - the "chance score" - was based on the probability of f i s h movement being random. For example, the probability of a f i s h moving from compartment 13 (middle compartment) to the next was one half. Then the probability of i t moving to the next was one quarter and so on to the end of the trough. A score was thus calculated for each f i s h , summed and then divided by the total number of f i s h , to give a mean. The formula was thus: 12 Z ni q-hX) i=l N T where n,- = number of fish per compartment 12 ^T = total number of fi s h moving in one direction = E n^ i=l Two scores were obtained from each experiment, a downstream score and an upstream score. The second score system tried was one in which a "rank number" was assigned to each compartment with the middle compartment being zero and the upstream, or downstream side, being numbered one to twelve consecutively with the end compartment being number twelve. The number of fi s h per com-partment was multiplied by i t s corresponding rank number, summed, and then divided by the total number of f i s h that had moved in one direction. To prevent negative scores, a constant of twelve was added to each score. The formula was: 12 Z (n ± xr) + 12 i=l 18 where IK = number of f i s h per compartment r = rank number of corresponding compartments = total number of fi s h moving in one direction A third score was also obtained by subtracting the downstream score from the upstream score and again adding a constant of 12 to prevent negative numbers. The third score tried was very similar to the second except t h a t % equalled the total number of fish used in that experimental test. That i s , i t gave the mean proportion of f i s h moving in any one direction. In a comparison of Hayman's analysis on the "chance score" and the f i r s t "ranked scores", i t was found that the same st a t i s t i c s were significant in both cases, but higher levels of significance were found in the "rank scores". This i s probably due to the fact that with the "rank score" system a higher value is given to the fi s h that move the farthest. In a comparison of the two "rank scores", exactly the same F-ratios were obtained in both cases for the net scores. The mean proportional rank system appeared to be the best to distinguish between a test with l i t t l e directional movement and a test with a large directional movement. Because a l l scoring systems were arbitrary and because there was basically l i t t l e difference between the systems, the proportional ranking system was used. Appendix 1 shows several hypothetical examples of scores for the upstream-downstream movement of fry. This scor-ing system would not distinguish between the situation where a l l fry remain-ed in the middle compartment (the score would be 12) and that where half of the fry moved into the downstream trap and the other half moved into the upstream trap (the score again would be 12). However, in no instance did a l l the f i s h in any one test remain in the centre release compartment. V RESULTS A. Behavioural Responses of Fry in the Experimental Channels The directional movement of the fry was largely dependent upon the 19 l i g h t as w i l l be shown l a t e r . When the l i g h t regime was changing from l i g h t to dark, the f r y would s t a r t to move upstream upon f i r s t being released. However, when the l i g h t was decreased to a very low l e v e l , upstream movement would cease and a f t e r two hours of t o t a l darkness movement would be predominately downstream. No observations were made of the f i s h i n the dark but i n daylight i f f r y moved passi v e l y downstream,' they would go from one compartment to the next. This was probably because of the staggered p o s i t i o n of the compartment openings and the " s w i r l i n g " action of the current which hindered any fur t h e r passive downstream movement. When moving upstream, the f r y had to put f o r t h extra e f f o r t to swim through the opening to the next compartment. However, they could hold p o s i t i o n i n areas of each compartment where the current was minimal. Most movement took place w i t h i n the f i r s t hour of the t e s t . The f i s h normally showed very l i t t l e "back-and-forth" movement through the troughs and i f they did, i t usually involved only two or three compartments. It was further observed during daylight that when the f r y reached the end compartments they would s t i l l t r y to move fa r t h e r upstream by bumping against the screen covering the i n l e t . B. S u r v i v a l The m o r t a l i t y associated with a l l crosses w i l l be dealt with i n greater d e t a i l i n a separate paper. However, the number of i n t r a -population crosses were reduced to s i x stocks as the Pennask Outlet eggs suffered 100% mortality. For the d i a l l e l crosses there was an unexplained high mortality i n the second cross. This was mainly i n the second r e p l i c a and was caused by the males (rows) of Pennask I n l e t , Thunder Creek, and Below Hihium stocks as w e l l as the females (columns) of Below Hihium. C. Performance Tests a) Pure stocks The t e s t s f o r the s i x pure stocks (Fig. 4) were run during darkness and daylight at low (4.7 ± .5 C) and high temperature (14.0 ± C). Analysis of variance shows that there was a highly s i g -n i f i c a n t d i f f e r e n c e between each of the three main e f f e c t s (P<.001) Fig. 4 Net scores for the behavioural tests for the current response of the pure stocks. Solid circles and bars represent means and ranges, respectively of 12 replicas. 21 of stock, l i g h t , and temperature (Table III). Some of the f i r s t order interaction terms were also significant when tested by the graphical method of Sokal and Rohlf (1969). The stocks by light (Fig. 5a), i.e. genotype-environmental interaction, was significant (P< .01). Here, the fry show a definite upstream response during daylight and a definite downstream response during darkness. Further, the stocks also change their intensity of movement in relation to each other between day and night tests. For example, Loon Outlet stock was f i f t h in order of upstream preference during the daylight, but was f i r s t in order of downstream preference at night. Pennask Inlet fish also show an interaction between stocks and light. The genotype-environmental interaction of the stocks at different temperatures is also highly significant (P< .001). In the experiment inlet fry had a greater upstream response during the day than the outlet stocks. During darkness, outlet fry showed a greater downstream response than inlet fry. This is directly opposite to f i e l d observations where inlet fry move downstream during the night and the outlet fry move upstream during the day. In other tests conducted on Loon Lake fry (Kelso MS, 1970) in the summer of 1969, there was a definite current response (Fig. 6). The chi-square test for independence for daylight tests was highly s i g n i f i -cant (P<..005). Both the Outlet stock and the Hihium stock showed a strong upstream preference while the Inlet stocks showed a downstream response in the daylight runs. However, here there was very l i t t l e difference between the day and night tests of the Outlet stock, whereas the Inlet stock showed greater downstream preference during the day than at night (Fig. 6.). However, the most important result was that a change in temperature did not cause the fry to reverse their direction but only caused a change in the intensity of their movement. The light by temperature interaction was not significant (Fig. 5) nor was the second order interaction of stock by light by temperature. The highest variance component was that of light - 74.5% of the total variance. Variance of the stocks was 3.2% while the stocks by light was 1.6% and the stocks by temperature was 5.1% (Table IV). 22 Table III. Analysis of variance on the net scores of the behavioural tests on the pure stocks using the three factors where A = stocks,B = light and C = temperature. Source d.f. M.S. F Main effects A = stocks 5 105.65 13.40 *** B = light 1 6881.32 872.85 *** C = temperature 1 281.34 35.69 *** A X B 5 31.75 4.03 ** A X C 5 85.78 10.88 *** B x C 1 4.83 <0n.s. A X B X C 5 11.28 1.43 n.s. Error 264 7.88 Total 287 F i g . 5 F i r s t order i n t e r a c t i o n s of the analysis of variance (Table III) on the pure stocks; s i g n i f i c a n c e indicated by the degree of crossing between l i n e s (see Sokal and Rohlf, 1969 p. 355). 24 Fig. 6 Upstream-downstream preference tests on three stocks of Loon Lake fry conducted in "Brannon-type" (Brannon, 1967) performance troughs during the summer of 1969. Water temperatures = 9.4C, velocity = 6.7 cm/sec, water source = Loon Creek Hatchery spring water. 25 Table IV. The expected mean squares and the variance components for the three-factor analysis of variance on the pure stocks, n = 12, a = 6, b = 2, c = 2. Expected Variance Percent Source mean square component variance A = stocks a 2 + nbcCTA 2.0369 3.2 B = light a 2 + nacCT? 47.7322 74.5 3 C = temperature a 2 + nabaf. 1.8990 3.0 A x B a 2 + nca^g 0.9943 1.6 A X C CT2 + nba 2 c 3.2456 5.1 B x C a 2 + naa 2 0.0 0.0 BC A x B x C a 2 + no2 0.2830 0.4 ABC Error a 2 7.8837 12.3 26 b) D i a l l e l crosses The scores for Blocks 1 and 2, together with, their means, are given in Appendix 2 for Crosses I and II at the three temperatures. (i) Hayman's analysis of the d i a l l e l tables As mentioned earlier, Hayman's analysis of the d i a l l e l cross computes four s t a t i s t i c s plus a breakdown of his (B) or dominance term into three further st a t i s t i c s (b^, b2» b^). The analysis of variance tables for a l l the tests are shown in Appendix 3 including the b^, b^, and b^ terms. The breakdown of the "B" term- w i l l not be considered herein. Expected mean squares for the "model for reciprocal effects" are given i n Table V, which best shows the genetic effects of a d i a l l e l cross. The score calculations for Hayman's analysis of the d i a l l e l cross were f i r s t divided into upstream and downstream movement and analysed separately. Then, these scores were combined by subtracting the downstream from the upstream to obtain a net score. An analysis was done on a l l three sets of scores. The results of each (not shown) were the same whether s p l i t or net scores were used with one exception at the medium temperature where dominance was shown (P<.05) for Cross II daylight and Cross I darkness, for s p l i t scores but not net scores. The net scores of the two replicas were also summed and an analysis done on these scores. The results of the two analyses on the net scores are summarized in Table VI. The additive effect (A), ie genetic variation among stocks was evident at a l l temperatures, but not necessarily i n both crosses or at both light conditions tested. Only in cross I for daylight tests was there additive genetic variance at high temperature (P<C.05) while at medium temperature the genetic variation was only significant during the darkness tests. However, when the crosses were summed there was additive genetic variance at both medium and low temperature tests. The only evidence of dominance (B term) was at low tempera-ture during darkness (P<.05) but this was not evident when the crosses were summed. 27 Table V. Model for expectations of mean squares of a n 2 d i a l l e l cross, when the parental lines are fixed, for Hayman's analysis of variance. Sour.ce Expected mean square A = parental lines B = genetic interaction C = average maternal effects D = reciprocal effects Error a 2 + 2ria 6 2 2 , 2n a s a + n-l a 2 + 2a r 3 a 3 + 2a r 2 28 .Table VI. Levels of significance for Hayman's analysis of variance of the d i a l l e l crosses for the net scores. "P" means the item was tested against the p ooled interaction ; mean square. (a) Net Movement Daylight Darkness •Temperature Cross I Cross II Cross I Cross II A n.s. n. s o n.s. B n.s. n.s. n.s. n.s. High C * p ** p n.s. ** p D n.s. n.s. n.s. n.s. A n.s. n.s. ** p *** p B n.s. n.s. n.s. n.s. Med. C n.s. n.s. n.s. n.s. D n.s. n.s. n.s. -k A n.s. v V * p * P •k-klt p B n.s. n.s. * * P Low C n.s. n.s. ** p •kick p D n.s. p * P ** (cont *d) 29 TableVI (cont'd) (b) Crosses Summed for Net Movement Temperature Daylight Darkness High Med. Low A n. s . B n.s. A * P B n.s. C n.s. n.s. n.s. C ** p ** p D n.s. * p n.s. n.s. D n.s. n.s. A *** p *** p B n.s. n.s. C n.s. *** p D * P * p 30 Maternal effects (C). were quite significant at high, temperature for daylight and darkness and at low-temperature for darkness, but not at the Intermediate temperature. At the medium temperature only in Cross II at darkness was there any reciprocal difference and this was in the " D " term, i e . those effects not ascribed to "C". At the low temperature during daylight there was no significance for maternal effects but the "D" term was significant for Cross II (P<.01). The intrapopulation v a r i a b i l i t y was further outlined i n the perceptage of the variance components of the d i a l l e l tables (Table VII). The additive component of variance ranged from 0 to 20 percent between the different tests, but also varied between crosses. The percent variance of the dominant effect was mostly zero except at low temperature. The error variance, ie the unexplained variance, was high which suggests that rearing and/or testing procedure could be an important factor. The additive and dominant effects of the genes were shown schematically with the use of a graph. When the score for a hybrid of an inlet and an outlet cross f e l l exactly half way between the scores of the two parent stocks, then the genes were considered to be completely additive and no dominance existed. If the hybrid score favoured one of the adult stocks then there was evidence of some dominance. However, i f the score f e l l somewhere outside the two parental stocks then this was considered "overdominance" (Falconer, 1960). The mean scores at high temperature for the leading diagonal (Fig. 2) of the d i a l l e l cross and some of the hybrids were plotted on a daylight-darkness graph to show the additive variation of the genes (Fig. 7). There was overdominance in a l l cases except the Loon Inlet by Loon Outlet cross where the Inlet stock was dominant over the Outlet stock. However, with hybrids of the Pennask Inlet/Pennask Outlet cross; the fry acted more like inlet fry with very l i t t l e movement during the daylight and a very strong downstream movement in the darkness. The same pattern also followed for the Thunder Inlet by Loon Outlet cross 31 Table VII* Percent of the variance components for Hayman's analysis of the d i a l l e l ; tables of the net scores. Pooled mean square used as error variance • (a) Net Movement Daylight Darkness Temperature Cross I Cross II Cross I Cross II A 4.37 0.47 .0 0.79 B 0 0 0 0 High C 39.66 50.87 11.55 58.77 D 6.10 11.88 0 0.79 E 49.86 36.78 88.45 39.65 A 5.49 2.63 11.02 19.57 B 0 20.20 17.27 4.22 Med. C 0 0 0 0 D 0.47 0 12.27 23.14 E 94.05 77.17 59.44 53.07 A 3.09 6.58 3.85 6.81 B 0 9.98 4.33 9.15 Low C 0. . 20.55 38.88 46.03 D 5.92 27.89 19.97 12.22 E 90.99 35.00 32.97 25.79 (cont 'd) 32 Table VII(cont'd) (b) Crosses Summed for Net Movement Temperature Daylight Darkness A 1.49 1.37 B 0 0 High C 54.38 51.63 D 10.21 14.38 E 33.91 32.62 A 9.50 22.15 B 5.35 4.59 Med. C 0 0 D 0 16.65 E 85.15 56.61 A 13.38 6.24 B 6.09 5.79 Low C 12.86 54.59 D 26.08 12.97 E 41.58 20.42 33 Fig. 7 The mean scores of both crosses for the "leading diagonal" (see Fig. 2) and some of the hybrids for the daylight and darkness tests conducted at the high temperature (17.5 ± 1.5C). Single symbols represent the self-crosses, paired symbols represent the hybrids. r LOON INLET • LOON OUTLET ~t~ HIHIUM CREEK CJ PENNASK OUTLET \ THUNDER INLET f 2 © PENNASK INLET Ad ©O - J 1 i 1 1 I 1 i i I I i yy I 2 3 4 5 6 7 8 9 10 II 12 24 DOWN STREAM ( —~\ MEAN SCORE DARKNESS 34 where the influence of the Inlet parent seemed to be the most dominant. This was also evident in hybrids from crosses of Hihium Creek stock with Loon Inlet and Loon Outlet stocks. The Loon Inlet/Hihium Creek hybrids showed l i t t l e movement during day-light and a strong downstream movement during darkness. Hybrids from the Loon Outlet/Hihium Creek cross showed a greater upstream movement in daylight and less downstream movement during darkness than the inlet fry. Fig. 7 shows a positive correlation with regards to upstream-downstream-movement. The three outlet stocks, Pennask Outlet, Loon Outlet and Hihium Creek,exhibited the greatest upstream move-ment during the daylight and the least downstream movement during the darkness. The inlet stocks showed the reverse. At the medium temperature there was overdominance in a l l cases (Fig. 8). However, here the Pennask Outlet self-crosses show greater downstream movement than the Pennask Inlet self-crosses. Also, Thunder Inlet shows a very high score during the day. ( i i ) The factorial analysis of variance From the results of Hayman's analysis of variance on the experimental current responses of the fry, i t would appear that the fish are reacting not only to genetic and maternal components, but also to environmental components as well. Wearden (1964) gives a model to test for maternal effects by using an analysis of variance between the rows and columns of the d i a l l e l crosses. One can further extend this analysis into a multi-factorial design and test for the environmental components of light and temperature as well. The results of such an analysis of variance on a l l the data i s summarized in Table VIII. Of the three main effects (males, females, and temperature), temperature is the most significant for a l l tests (P< .001). The male component i s only significant during darkness.(P< .05) while the female component is significant in a l l cases except cross I Darkness. The graphical interpretation of the male by female interaction shows that the female components of the d i a l l e l cross had a slightly 35 Fig. 8 The mean scores of both crosses for the "leading diagonal" and some of the hybrids for the daylight and darkness tests conducted at the medium temperature (9.7 ± .5C). Single symbols represent the self-crosses, paired symbols represent the hybrids. I-X CD _J >-24 23 22 21 20 19 18 < 17 UJ UJ £ or co i6 O OL o => co < LU 15 14 I-13 12 I i THUNDER INLET LOON OUTLET o +• -J" HIHIUM CREEK m(ff- G LOON INLET SLPENNASK OUTLET A • ©O PENNASK INLET 0 1 2 3 4 5 6 7 8 9 10 11 h-• DOWN STREAM 12 24 -I H MEAN SCORE DARKNESS Table VIII Three -factor analysis of variance table on the net scores where A = males, B = females, C = temperature for the daylight and darkness runs for both Cross I and Cross I I . Source Cross I Daylight Cross I I Daylight Cross I Darkness Cross I I Darkness d.f. M.S. M.S. M.S. M.S. Main effects A = males 6 B = females 6 C = temperature 2 20.9539 1.81 n.s. 37.5122 2.04 n.s. 28.9789 2.50 * 39.6986 2.16 * 20.4406 2.39 * 45.3519 2.80 * 15.7645 1.84 n.s. 94.1453 5.81 *** 695.0153 59.98 *** 162.5654 8.84 *** 189.2228 22.12 *** 256.5955 15.84 *** Interactions A * B A X C B X C 36 19.4585 1.68 ** 30.6723 1.67 * 12 12.6842 1.09 n.s. 43.6188 2.37 ** 12 16.4793 1.42 n.s. 31.5760 1.72 n.s. 8.6791 1.01 n.s. 35.4247 2.19 *** 13.2756 1.55 n.s. 59.9597 3.70 *** 9.2704 1.08 n.s. 45.1004 2.78 ** A x B x C 72 6.8715 0.59 n.s. 23.7895 1.29 n.s. 10.4396 1.22 n.s. 15.6011 0.96 n.s. Error 147 11.5866 18.3953 8.5547 16.2000 Total 293 37 higher score than the male components, (Fig. 9), although there were some exceptions. These were Loon Inlet and Pennask Outlet, and i t was these stocks that showed the greatest degree of inter-action. The two genotype by environmental interactions, i.e. male by temperature and female by temperature, are shown in Figures 10 and 11. Although temperature was highly significant (P< .001) in the main effects i t did not appear so important in the inter-actions. For the male by temperature, i t was significant for Cross II. This was due mainly to the Thunder Inlet stock at the medium temperature during daylight arid the Pennask Outlet and Below Hihium stocks during darkness. For the female by tempera-ture interaction, only cross II Darkness was significant (P<.01). Again, this was due to Pennask Outlet and Below Hihium stocks. This shows intrapopulation v a r i a b i l i t y as well as interpopulation v a r i a b i l i t y . The variance components of the analysis again show that the environmental components of temperature i n the interactions was quite low (Table i x ) . Only for the interaction of males by tem-perature for cross II darkness does the variance reach 10% of the total variance. Most of the variance in the analyses was due to the error term, which again indicates that rearing and/or testing procedure, ie environmental effects, could be important. As with Hayman's analysis, the analysis of variance further points out that there are genetic differences among the parents. However, two very important features are shown in the graphical interpretations of the f i r s t order interactions. F i r s t , the only environmental component that causes an actual change in direction i s light. In the daylight, the movement is predominately upstream while in the darkness i t is predominately downstream. The second important feature is that the three different temper-atures do not cause a change of direction in the fish movement but only a change in the intensity of the movement either upstream or downstream. The results of the leading diagonal was plotted on a high temperature by low temperature graph (Fig. 12). For the daylight tests the order of the upstream migrants and the downstream migrants are as one would excpect with the Inlet fry showing the 38 Fig. 9 First order interactions of male (A) by female (B) in the factorial analysis of males by females by temperature. r 2 3 2tf 21 2C|-19 18 1 '7 UJ 0-UJ or o o </> LU 15 14 13 12 II 10 9 8 2 < to 7 § 6 M A L E X F E M A L E DAY LIGHT DARKNESS (o) CROSS I P= * * (b) CROSS 11 P= * (c) CROSS I P— n-s 1 PENNASK INLET 2 LOON INLET 3 THUNDER INLET 4 PENNASK OUTLET A 5 LOON OUTLET 6 BELOW HIHIUM 7 7 HIHIUM 5 a i 6 3 2 (d) CROSS II l»6 MALE FEMALE MALE FEMALE MALE FEMALE MALE FEMALE 39 Fig. 10 First order interactions of male (A) by temperature (C) in the factorial analyses of males by females by temperature. LU or o o co - 2 3 22 21 2 0 19 18 < 1 7 UJ <r i - 16 VI a. => 15 MALE X TEMPERATURE D A Y L I G H T (o) C R O S S I (b) C R O S S II P - n .s . P = * * LOW D A R K N E S S (c) C R O S S I (d) C R O S S II P = n . s . P = * * * MEDIUM MEDIUM HIGH 10 2 < UJ cc 5 o o oi S T O C K P E N N A S K I N L E T L O O N I N L E T T H U N D E R I N L E T P E N N A S K O U T L E T L 0 0 N O U T L E T B E L O W H IH IUM H I H I U M LOW LOW MEDIUM MEDIUM HIGH HIGH I 2 3 4 5 6 7 I 2 3 4 5 6 7 I 2 3 4 5 6 7 I 2 3 4 5 6 7 MALE STOCK 40 Fig. 11 First order interactions of female (B) by temperature (C) i n the factorial analyses of males by females by temperature. F E M A L E STOCK Table IX. Expected mean squares for three-factor analysis of variance, fixed model, and the variance components for the four analyses of Cross I and II for daylight and darkness, n = 2, a = 7, b = 7, c = 3. Variance component Percent variance Source Expected mean square Cross I Daylight Cross II Daylight Cross I Darkness Cross II Darkness Cross I Daylight Cross II Daylight Cross I Darkness Cross II Darkness A = males a 2 + nbccj^ 0.2230 0.4552 0.2830 0.6941 1.07 1.61 2.32 2.35 B = females a 2 + nacti 2 a 0.4141 0.5072 0.1717 1.8558 1.98 1.79 1.41 6.27 C = temperature a 2 + nabtj2 6.9738 1.4711 1.8436 2.4530 33.31 5.20 15.11 8.29 A X B a s • *>2 + n C C TAB 1.3120 2.0462 0.0207 3.2041 6.27 7.23 0.17 10.83 A X C a 2 + n b*AC 0.0784 1.8017 0.3372 3.1257 .37 6.36 2.76 10.56 B X C a 2 • * 2 + n a C TBC 0.3495 0.9415 0.0511 2.0643 1.67 3.32 0.42 6.97 A X B x C a 2 + n C TABC <0 2.6971 0.9425 <0 0 9.53 7.72 0 Error a 2 11.5866 18.3953 8.5547 16.2000 55.34 64.97 70.10 54.74 42 Fig. 12 The mean scores of both crosses for the "leading diagonal" in a linear regression of high temperature and low temperature for daylight and for darkness. Question mark_means i t i s uncertain i f the stock should be called an upstream or a downstream stock. 4 6 • DOWN STREAM -18 20 UP STREAM •— 22 24 MEAN NET SCORE LOW TEMPERATURE 43 l e a s t movement and the. Outlet f r y showing the greatest upstream movement. The same applies f o r the darkness tests except f o r the Pennask I n l e t tests at the low temperature where there was very l i t t l e movement. Question marks appear beside the Hihium (7) and Below Hihium (6) stocks because one can only postulate as to t h e i r behaviour. I t i s expected . the Hihium f r y would move upstream during the daylight and downstream during darkness. As for the Below Hihium f r y , i t i s believed that there are some resident stock spawning i n t h i s area and I t Is d i f f i c u l t to speculate as to how t h e i r progeny would behave i n the experimental channels. VI DISCUSSION Conditions of the i n l e t and o u t l e t streams of Loon Lake were studied i n d e t a i l by Northcote (1962) and observed by myself i n the summer of 1969. Northcote found that downstream movement of rainbow trout f r y occurred i n both I n l e t and Hihium Creeks where the water temperature r a r e l y exceeded 13 C. This migration took place almost e n t i r e l y at night when i l l u m i n a t i o n f e l l below 0.01 foot-candles. He reported some occasional downstream move-ment i n the Outlet Creek when the water temperature was >14 C f o r several days. Further, he found that the Outlet f r y maintained p o s i t i o n at night, but only when water temperature was >14 C. The upstream movement of the f r y occurred only i n the Outlet Creek where the summer water temperature was >15 C. I t i s also known that the I n l e t f r y move downstream in t o the lake s h o r t l y a f t e r emergence from the gravel, while the Outlet f r y remain i n the creek f o r one to two months or even up to one or two years before migrating to the lake. The d i e l movement of sockeye salmon i s also s i m i l a r to rainbow tr o u t , where the upstream movement i s almost e n t i r e l y i n the daylight and the downstream movement i s predominately at night (Brannon 1967; McCart 1967). McDonald (1960) also reports that pink, coho and chum f r y movement downstream was nocturnal and rather p r e c i s e l y regulated by l i g h t and i t s changes i n i n t e n s i t y . From more recent studies on sockeye salmon and other stream systems con-44 taining trout, i t would appear that there are innate as well as environ-mental controls governing the migratory behaviour of rainbow trout. The results obtained in this study further support this hypothesis. However, the experimental results here suggest that the marked differences of water temperatures between the two types of stream systems are not one of the major controls affecting the lakeward migration of the young trout. A. Genetic Effects The Mendelian method of studying genetic traits involves crossing known genotypes differing i n phenotype and arriving at the f i r s t (F^) and second (F^) f i l i a l generations of offspring and then backcrossing these to the parent strains. However, Broadhurst (1967) points out that prior to 1956 there were only four cases where this type of analysis was applied to behavioural characteristics. Further, this type of analysis i s not very feasible for long lived species. Therefore, the d i a l l e l cross i s believed to be the best way to determine whether there are genetic differences between families when one can only deal with one generation of progeny (Broadhurst, 1967). Both s t a t i s t i c a l methods used in the analyses of the data herein show that there is a genetic difference between the behavioural current responses of the seven stocks chosen. One should expect from f i e l d observations that i f there are genetic differences between the Inlet and Outlet fry that when they are tested together under the same light conditions, the Inlet fry should hold during daylight while the Outlet fry should move upstream. The reverse should happen during darkness where the Outlet fry should hold and the Inlet fry move downward. However, in Loon Outlet Creek no fry move up to the lake immediately after hatching. Some remain in the creek for two to three months while others remain in the creek for one to two years. Thus, at the age at which the fry from the d i a l l e l cross were tested they should remain in the test apparatus for both daylight and darkness tests. However, Slaney (MS 1972 and personal communication) found that at very low food levels a large number of fry moved out of his test channel, while at high food levels most of the fry remained. Of the inlet fry that moved out of the testing channel at the low food level, 70% moved downstream. 45 At the high food level 70% moved upstream, but this was a lesser number of fi s h than at the low food level. For the outlet fry, of those moving out of the test channel 50% went upstream and 50% went downstream at both food levels. This suggests that the absence of food also plays a key role in the start of migration of the young fry. No food was present i n the current response channels and this might explain why the Outlet stocks moved upstream during the day. If this explanation is true, then Figure 12 shows that the fry did behave as expected in the experimental troughs with some exceptions. •Variations between replicas can pa r t i a l l y be explained by Lindsey et a l . (1959). In their study of adult rainbow trout they found that some mixing of the two populations did occur. Thus, there is always the possibility that the adults collected from the Outlet could have included some f i s h originating from the inlet or vice versa. Thus, at no time would an Inlet or an Outlet stock show 100% movement in the required direction. Lindsey et a l . (1959) reported that homing was 94% accurate for both streams. However, the Inlet usually contains two to three times more f i s h than the Outlet so the chance of picking up an Outlet adult in the Inlet i s two to three times less than in the Outlet. The fact that the Outlet fry do not usually move upstream u n t i l they are older than those used in the tests, might also cause some v a r i a b i l i t y in these tests. It i s very d i f f i c u l t to say whether there is a greater intrapopulation v a r i a b i l i t y within the outlet stock than within the inlet stock. However, the graphical interpretation of the male by female interactions did show that the Pennask Outlet male-female crosses had the greatest amount of inter-action for both replications. Also, the Loon Inlet male-female crosses of the second replica showed a large interaction. Further, one would expect that there would not always be a 100% movement in one direction and that there should always be some variation in the population. Thus, i f a disaster occurred in one stream there would always be a small proportion of the population l e f t to carry on the population. However, the most important behavioural aspect of these tests is that the fry were behaving in the appropriate way to light and to dark 46 tests as to what had been observed i n the f i e l d . Because there was more upstream movement by the Outlet stocks in daylight tests and more downstream movement by the Inlet stocks i n darkness tests, the evidence for genetic differences between the stocks is further strengthened. Evidence for genetic differences between stocks is also seen when one examines the crosses of the inlet stocks with the outlet stocks. Figures 7 and 8 show overdominance as well as additive genetic effects. In most cases the inlet stocks appear to be slightly dominant over the outlet stocks especially for Loon Inlet at the high temperature. The crosses of the Hihium stock with the Inlet and Outlet stocks also show that the Inlet parent was partially dominant with respect to current response. In a similar study by Calaprice (1972b) i t was found that there was additive genetic variation between sockeye stocks of the Babine Lake system. The s t a t i s t i c a l analyses on the tests performed with pure stocks also show that there i s a difference between the stocks. However, these differences do not seem consistent with those apparent in the f i e l d . That i s , while there is a marked difference between daylight and darkness tests, the Inlet stocks showed more upstream movement in the day and the least movement at night. Precautions were taken to ensure that there was no mixing of the stocks, but the absolute possibility of this happening cannot be ruled out. One possible explanation of the current response of the pure stocks and the intrapopulation v a r i a b i l i t y of the d i a l l e l cross could arise from three phenotypic forms of l i v e r lactate dehydrogenase (L.D.H.) that exist in the fish of the two lakes systems. Northcote, et al.- (1970) found that there were three phenotypic forms of L.D.H. in stream populations of rainbow trout from above and below a waterfall. These consisted of two homozygous strains (CC and C'C') and a heterozygous strain (CC 1). It 47 has since been shown that the CC strain has the a b i l i t y to r i d i t s e l f of l a c t i c acid i n the muscle tissues four to five times faster than the other homozygous strain (H. Tsuyuki, personal communication). This allows the above f a l l s population, CC, to remain in the faster flowing water for a much longer time period. In preliminary studies (unpublished data, H. Tsuyuki) i t was found that the three strains existed in the Loon and Pennask Lake systems. Tests were conducted on Loon Inlet fry with the two homozygous strains (CC and C'C') of L.D.H. (Fig. 13). The CC strain showed more upstream movement during the day while the C'C' showed greater downstream move-ment at night (P<C.001). Thus, the phenotypic form of the adults could have greatly influenced the current response of the offspring used to test the pure stocks. Unfortunately the L.D.H. types of the parents used in the pure stocks are not known but an analyses was done on the adults of the d i a l l e l cross (Table X) and from this i t i s possible that there could have been a very high percentage of CC strains in the Inlet stocks. The interpopulation v a r i a b i l i t y i s not significant (P>.05), but there is a' large intrapopulation va r i a b i l i t y (P<.01). The Pennask system for both the Inlet and Outlet have a high percentage of the CC strain. However, the stocks taken from the Outlet system of Loon Lake have a larger percentage of the C'C' than the CC. Furthermore, because there i s a large v a r i a b i l i t y in the number of L.D.H. strains between replicas of the d i a l l e l cross, this could also pa r t i a l l y explain the intrapopulation v a r i a b i l i t y in the current response of the d i a l l e l cross. 48 Fig. 13 Net score of the current response of Loon Inlet fry which contain two separate phenotypic forms of l i v e r lactate dehydrogenase. Tests conducted in 25-compartment performance channels. Circle and bar represent mean and ranges, respectively of 12 replicas.. Data provided by T.G. Northcote; to be published elsewhere. UJ o CJ CO f-UJ T 24 22 20 I 18 rr to OL 3 16 14 - - 12 10 < UJ r- 6 r-CO z s 4 h Q -L 0 D A Y L I G H T - D A R K N E S S CC CC LOON INLET STOCK 49 Table X. The percent of the adults used in the d i a l l e l cross which contain the three types of l i v e r lactate dehydrogenase (L.D.H.). See text for explanation of the three types of L.D.H. Stock Cross Number % CC % CC % C'C Pennask Inlet I 16 56 44 II 11 55 36 9 mean 55.5 40 4.5 Loon Inlet I 14 7 64 29 II 15 33 67 -mean 20 65.5 14.5 Thunder Creek I 12 50 17 33 II 13 15 39 46 mean 32.5 28 39.5 Pennask Outlet I 13 46 46 8 II 14 64 29 7 mean 55 37.5 7.5 Loon Outlet . I 13 15 39 46 II 13 31 46 23 mean 23 42.5 34.5 Below Hihium I 10 _ 50 50 II 20 10 35 55 mean 5 42.5 52.5 Hihium Creek I 12 _ 58 42 II 21 14 62 24 mean 7 60 33 50 B. Maternal Effects Very l i t t l e can be said about maternal effects other than that the analysis shows they do exist. One possible explanation could be that an extra amount of cytoplasm was contributed by some females to their eggs, and thus, the alevins would have a larger yolk sac. This might allow some fry to be stronger than others in their swimming a b i l i t i e s . Calaprice (1972b) found in his study of sockeye salmon that there was no maternal influence when he tested the fry for current response. However, he did find (Calaprice, 1972a) that maternal effects were directly related to the survival of the young fry. One reason was the presence of parasitic nematodes in the females. In this study of rainbow trout, there was no evidence of internal parasites in the Loon Lake females, but both stocks of females from Pennask Lake carried a very large number in the body cavity. Calaprice also states that maternal effects could be a possible mechanism for decreasing the reproductive potential of a population and thereby affecting regulation. Eisen (1967) points out that maternal effects can mask genetic effects. However, one would need several generations of trout in order to test for this. C. Temperature Effects The most obvious environmental differences between the inlet and outlet streams of both Pennask Lake and Loon Lake are the differences in water temperature. The experimental results here, however, suggest that different water temperatures are not the main influence which causes the appropriate current response of the two types of stocks. Further, Brannon (1967) in his study of sockeye salmon, reports in his f i e l d observations that there is only a 2C difference between the water temperatures of the Chilko River (upstream races) and the Stellako 51 River (downstream races). In his experimental tests he found that temperature had no effect on directional preference. In these experiments the different water temperatures only caused an intensity change in directional movement. Furthermore, this intensity change was the opposite to what one would expect from f i e l d observations. That i s , there was even less upstream movement at the high temperature than at the low temperature for both daylight and darkness tests. This was also evident for the tests conducted on the pure stocks. However, when one examines the results as shown in Fig. 10 and 11, at high temperature i t i s the inlet stocks that moved the least amount during daylight. At the low temperature during the darkness tests the inlet stock moved the farthest, with the exception of the Pennask Inlet stock. Here there could have been some mixing of the two Pennask spawning stocks as there are some differences between the male by female interaction as shown i n Fig. 9d. Thus, the different water temperatures'may have some influence on the migration behaviour of the fry. If water temperature was playing a key role in the migration of inlet and outlet fry, one would expect they would have a high upstream score of twenty-four during the high temperature and a very high down-stream score of near zero at the low temperature. This is on the assump-tion that the early outlet fry move upstream because of very low food levels. This would give a slope of zero on a high temperature score by low temperature score graph with the line running par a l l e l to the Y-axis. If temperature was only causing an intensity change, then during the daylight, the Outlet stock would have a score of near twenty-four while the Inlet stock would have a value of twelve or slightly higher due to the mixing of stocks and the slight temperature effect. This would then give a positive correlation on the temperature graph as is shown in 52 Fig. 12. It i s important to mention that this influence of temperature i s not the same as Northcote's further conclusion that sharp rises in water temperature are associated with upstream movement. This has been observed in several other lakes as well (Northcote 1969). Raleigh and Chapman (1971) found that experimental tests at different temperatures with cutthroat trout fry altered the ratio of outlet fry moving upstream and downstream, but did not alter the direction of move-ment of inlet fry. Raleigh (1971) found i n a study with sockeye salmon, that temperature changes had a greater effect on outlet stocks than inlet stocks, but again, this was only an intensity change and not a directional change. He further points out that temperatures, such as cold fluctuations in the outlet streams, only delay upstream migration and do not prevent the fry from eventually reaching the lake. D. Other Possible Effects a) Water sources In Northcote's (1962) study on Loon Lake an experiment was conducted using Loon Outlet and Hihium Creek water to test for the fry's behavioural response to temperature differences. He found that there was considerably more downstream movement in the cooler Hihium water than the warmer Outlet water where the movement was mainly upstream. Brannon (1967) in a similar test with sockeye fry at Cultus Lake, although his results were similar to Northcote's findings, concluded that the cue e l i c i t i n g the upstream response was in the lake water and was not due to temperature differences. Raleigh (1971) states that sockeye salmon fry can distinguish between sources of water and obtain directional cues, but that this mechanism 53 is poorly understood. b) Other environmental effects As stated earlier, the environmental component of variance consists of maternal effects as well as any effect that the animal's surroundings may have. In a dial le l cross the rearing facil it ies as well as the testing procedure are thus included as being part of the environmental component. In both Hayman's analysis and the factorial analysis of variance the percent error variance was very high (Table VII. and IX). This suggests that rearing and/or testing procedure could also be important, although there could also be unknown factors that caused the error variance to be so high. VII CONCLUSIONS (1) Experimental results of the progeny of a dial le l cross between seven inlet and outlet spawning stocks of Loon Lake and Pennask Lake, British Columbia, indicate that there are genetic differences in behavioural responses to current between the stocks. (2) These differences are such that basically the inlet stocks hold during daylight tests and move downstream during darkness tests while the outlet stocks move upstream during daylight tests and hold during darkness tests. (3) The most obvious differences between the inlet and outlet streams is the water temperature difference with cool inlet water and warm outlet water. The experiments conducted in this study show that different water temperatures do not cause a direction change in fry's current response, but 54 only an intensity change in its movement. However, at the temperature of 18 C the outlet fish did show the greatest upstream movement during the day-light and the least downstream movement during the darkness. (4) Although there is a large intrapopulation variability, genetic differences between populations and light intensity appear to be the most important mechansism controlling migration while temperature differences between streams only play a minor role. (5) Other mechanisms that may operate in the control of migration are water quality and source, the absence or presence of food, and the heritable trait of liver lactate dehydrogenase and its ability to dissipate lactic acid in the muscle tissue. 55 BIBLIOGRAPHY Brannon, E .L. 1967. Genetic control of migrating behaviour of newly emerged sockeye salmon fry. Int. Pac. Salmon Fish. Comm. Progr. Rep. 16: 31 p. Broadhurst, P.L. 1967. An introduction to the dial lel cross, p. 287-304. In Behaviour genetic analysis. Series in Psychology. McGraw-H i l l Book Co., New York, N.Y. Calaprice, J.R. 1972a. Heritable variation in five populations of sockeye salmon, Oncorhynchus nerka. I. Early migration. (Submitted for pub-lication.) Calaprice, J.R. 1972b. Heritable variation among populations of sockeye salmon, Oncorhynchus nerka. III. Differences in the migratory behaviour of fry in a current. (Submitted for publication.) Eisen, E . J . 1967. Mating designs for estimating direct and maternal genetic variances and direct maternal genetic covariances. Can. J . Genet. Cytol. 9: 13-22. Falconer, D.S. 1960. Introduction to quantitative genetics. The Ronald Press Co. N.Y. 365 p. Harden-Jones, F.R. 1968. Fish migration. Edward Arnold (Publishers) Ltd . , London, W.I. 325 p. Hayman, B.I. 1954a. The theory and analysis of the dial le l crosses. Genetics 39: 789-809. Hayman, B.I. 1954b. The analysis of variance of dial le l tables. Biometrics 10: 235-244. Hoar, W.S. 1953. Control and timing of fish migration. Biol . Rev. 28: 437-452. Kearsey, M.J. 1965. Biometrical analysis of a random mating population: A comparison of five experimental designs. Heredity 20: 205-235. Kelso, B.W. 1970. The upstream-downstream migration of rainbow trout in inlet and outlet streams of Loon Lake, British Columbia. Directed studies under T.G. Northcote, Dept. of Animal Resource Ecology, University of British Columbia. Lindsey, C C , T.G. Northcote, and G.F. Hartman. 1959. Homing of rainbow trout of inlet and outlet spawning streams at Loon Lake, British Columbia. J . Fish. Res. Bd. Canada 16: 695-719. 56 McCart, P. 1967. Behaviour and ecology of sockeye salmon fry in the Babine River. J . Fish. Res. Bd. Canada 24: 375-428. McDonald, J . 1960. The behaviour of Pacific salmon fry during their downstream migration to freshwater and saltwater nursery area. J . Fish. Res. Bd. Canada 17: 655-676. Mather, K. 1971. On biometrical genetics. Heredity 26: 349-364. Mather, K. , and J . L . Jinks. 1971. Biometrical genetics. 2nd ed. Chapman and Hall Ltd . , London. 382 p. Northcote, T.G. 1962. Migratory behaviour of juvenile rainbow trout, Salmo  gairdneri, in outlet and inlet streams of Loon Lake, British Columbia. J . Fish. Res. Bd. Canada 19: 201-270. Northcote, T.G. 1969. Patterns and mechanisms in the lakeward migratory behaviour of juvenile trout, p. 181-204. In T.G. Northcote (ed.) Salmon and trout in streams. H.R. McMillan Lectures in Fisheries, University of British Columbia, Vancouver, B.C. "~ Northcote, T . G . , S.N. Williscroft, and H. Tsuyuki. 1970. Meristic and lactate dehydrogenase genotype differences in stream populations.of rainbow trout below and above waterfall. J . Fish. Res. Bd. Canada 27: 1987-1995. Raleigh, R.F. 1967. Genetic control in the lakeward migrations of sockeye salmon (Oncorhynchus nerka) fry. J . Fish. Res. Bd. Canada 24: 2613-2622. Raleigh, R.F. 1971. Innate control of migrations of salmon and trout fry from natal gravels to rearing areas. Ecology, Vol. 52, No. 2: 291-297. Raleigh, R.F. , and D.W. Chapman. 1971. Genetic control in lakeward migra-tions of cutthroat trout fry. Trans. Amer. Fish. Soc. No. 1: 33-40. Slaney, P.A. 1972. Effects of prey abundance on distribution, density, and territorial behaviour of young rainbow trout in streams. M. Sc. thesis, Univ. of British Columbia. 74 p. Sokal, R.R., and F . J . Rohlf. 1969. Biometry. W.H. Freeman and Company, San Francisco. 776 p. Wearden, S. 1964. Alternative analysis of the dial le l cross. Heredity 19: 669-680. Appendix 1. Worked examples showing maximum, minimum downstream-upstream movement of fish in twenty-two hypothetical tests using the formula: I(n+r)/Nr + 12 where n = number of fish per compartment, r = rank number, NT = total number bf fish used in the test. 12 11 10 9 10 11 12 Upstream score Downstream score Compartment 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 1 0 2 1 3 2 4 3 5 4 6 5 7 6 8 7 91 8 10 9 11 10 12 11 13 12 14 13 15 14 16 15 17 16 18 17 19 18 20 19 21 20 22 0 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 4 12.000 12.600 13.200 13.800 14.400 15.000 15.600 16.200 16.800 17.400 18.000 18.600 19.200 19.800 20.400 21.000 21.600 22.200 22.800 23.400 24.000 12.833 24.000 23.400 22.800 22.200 21.600 21.000 20.400 19.800 19.200 18.600 18.000 17.400 16.800 16.200 15.600 15.000 14.400 13.800 13.200 12.600 12.000 17.944 58 Appendix 2. The behavioral scores for the d i a l l e l cross (for Blocks 1 and 2, Cross I and II).togehter with their means at.the three_.temperatures (17.5 i 1.5°C; 9.7 ± 0.5°C; 5.0 ± 1.5°C). NET D I A L L E L SCORES CROSS I OAYLI G M T , H I G H T E M P E R A T U R E F E M A L E 1 3 . 8 0 0 1 7 . 8 5 0 2 1 6 . 5 00 7.349 1 3 . 3 9 9 3 . 761 4 4.450 j .700 5 1 5 . 2 5 0 1 3 . 3 0 0 6 14.350 5.099 1 4 . 7 5 0 1 3 . 0 50 1 5 . 8 2 5 1 ? . 1 7 5 1 1 . 0 8 0 6 . 0 7 5 1 4 . 5 2 5 9.725 1 3 .900 1 2 . 0 5 0 1 4 . 4 5 0 1 3 . 2 50 1 9 . 6 5 0 1 1 . 4 7 3 1 5 . 5 6 1 11.750 1 3 . 4 4 9 12.600 1 2 . 3 5 0 1 0 . 3 5 0 1 1 . 3 5 9 1 6 . 5 0 0 1 3 . 9 4 9 1 5 . 2 2 5 1 3 . 0 0 0 1 9 . 1 5 0 1 6 . H 7 5 1 1 . 3 5 0 1 8 . 4 0 0 1 4 . 8 7 5 1 4 . 3 0 0 1 1 . 9 0 0 1 3 . 1 0 0 1 7 .300 2 0 . 7 0 0 19.250 7.699 1 3 . ^ 5 0 10 . q 2 5 1 4 . 3 5 0 1 6 . 4 0 0 1 5 . 3 7 5 1 4 . 6 5 0 11 . 9 5 0 1 3 . 3 0 0 1 6 . 4 5 0 1 5 . 2 5 0 1 6 . 8 5 0 9 . 4 0 0 1 4 . 4 5 0 1 1 . 9 2 5 1 7 . 9 0 0 1 6 . 1 5 0 1 7 . 0 2 5 1 4 . 7 5 0 1 5 . 7 0 0 1 5 . 2 2 5 1 8 . 3 50 2 0 . 1 0 0 1 9 . 2 2 5 1 4 . 6 5 0 16.H00 1 5 . 3 2 5 1 2 . 7 6 4 1 3 . 6 5 0 1 3 . 2 0 7 1 7 . 3 0 0 1 6 . 0 5 0 1 6 . 6 7 5 1 4 . 6 0 0 1 4 . 4 0 0 1 4 . 5 0 0 6 . 9 5 0 2 2 . 7 0 0 1 1 . 1 9 9 1 2 . 4 4 9 8. 3 99 1 9 . 7 5 0 9 . 5 5 0 1 6 . 5 0 0 2 0 . 100 2 0 . 0 0 0 1 6 . 9 5 0 1 9 . 7 0 0 2 0 . 8 0 0 14.649 1 4 . 3 2 5 1 1 . 8 2 4 1 4 .07 5 1 3 . 0 2 5 2 0 . 0 5 0 1 8 . 3 2 5 1 7 . 7 2 5 1 2 . 2 0 0 1 3 . 1 0 0 1 2 . 6 5 0 1 1 . B O O 1 5 . 5 5 0 1 3 . 6 7 5 2 2 . 3 5 0 1 7 . 3 5 0 1 9 . 8 5 0 1 7 . 3 0 0 e . 7 0 0 1 3 . 0 0 0 1 4 . 4 5 0 1 6 . 6 5 0 1 5 . 5 5 0 1 5 . ^ 0 0 1 6 . 2 0 0 1 6 . 0 5 0 1 0 . 8 0 0 8. 5 0 0 9 . 6 5 0 7 . 7 5 0 1 5 . 2 0 0 «.526 12 . 9 0 0 1 5 . 8 5 0 1 7 . 2 0 0 14.100 14.684 1 7 . 2 5 0 16 . 550 1 5 . 7 0 5 2 2 . ^ 0 0 1 3 . 100 1 7 . 2 00 11.475 1 0 . 7 1 3 1 6 . 5 2 5 1 4 . 3 9 2 1 6 . 9 0 0 1 8 . 9 5 2 15.150 59 Appendix 2 (Cont'd) NET DIALLEL SCORES CROSS II DAYLIGHT,HIGH TEMPERATURE F E M A L E 16.700 12.400 14.55C 1?.750 l B . o n o 1*.375 16.950 1-8.350 17.650 4 16.400 13.300 14.050 •5 11 .850 18.950 15.400 6 14.^00 16.250 15.125 7 1.9.450 21.050 20.250 18.550 1 9.950 19.251 12.200 16.350 14.275 2 0.2 00 16.050 13.125 14.750 18. 150 16.450 18.150 23.350 20.750 24.000 24.r.00 24.^00 11.700 22.350 17.C25 22.OOC 20.000 9. 399 1R.250 13.700 24.000 12.200 12.833 13. 700 0.000 0.^00 0.000 16.8 50 17.333 M A L E 21.000 17.050 6. 000 11.525 1 3.825 18.950 10.350 19.150 18.8 50 12.150 18.900 15.525 12.516 13.550 21 .050 17.300 6.850 13.^00 18.500 16.200 0.000 19. --00 l . Q 0 0 10.500 17.091 19.800 2 3.950 21.875 14.700 14.850 14.775 13.800 11.149 12.475 19.250 23.400 21.325 14.950 11.666 13.308 22 .100 17.800 19.950 2.599 23.500 13.050 19.350 16.200 17.775 15.444 13.000 14.222 20.375 9. 600 14.987 20.500 24.0?C 22.250 18.062 12.375 15.218 15.550 21 .000 18.275 23.^00 11.^00 17.500 22.500 23.333 22.916 20.250 18.05G 19.150 9.000 16.050 12.525 18.7 00 15.850 17.275 11 .050 19.000 15.025 15.750 18.050 16.900 13.875 19.571 16.723 21.450 22.550 22.000 60 Appendix 2 (Cont'd.) NET DIALLEL SCORES CROSS I DARKNESS,HIGH TEMPERATURE F E M A L E 1 2 3 4 . 5 6 7 3.700 1.849 3.299 5.150 7.349 0.600 4.750 11.100 6.000 8.000 4.950 7.000 2.666 2.949 7.400 3.925 5.650 5.050 7.175 1.633 3.850 1.000 8.899 3.900 4.150 7.899 1.599 9.600 15.600 4.800 7.200 1.200 7.250 1.250 0.000 8.300 6.849 5.550 2.675 7.575 1.425 4.800 3 10.050 2.399 3.350 4.800 5.800 1.200 20.500 5.000 0.000 9.399 2.399 4.550 8.949 3.500 M 7.525 1.200 6.375 3.599 5.175 5.075 12.000 A 4 8.300 9.850 13.200 9.550 10.000 6.294 11.300 L 8.149 3.500 2.950 6.850 3.399 4.099 5.700 E 8.225 6.675 8.075 8.200 6.700 5.197 8.500 1.899 1.200 2.399 3.950 10.350 6.149 4.750 12.900 4.550 5.649 5.250 10.000 7.300 4.650 7.400 2.875 4.024 4.600 10.175 6.725 4.700 3.850 8.749 6.800 4.900 2.100 7.750 2.799 6.300 8.150 9.350 2.399 4.950 7.599 2.950 5.075 8.450 8.075 3.650 3.525 7.675 2.875 3.350 0.300 4.950 2.700 6.000 3.588 2.000 3.600 1.149 9.600 3.650 3.699 12.538 3.650 3.475 0.725 7.275 3.175 4.850 8.063 2.825 61 Appendix 2 (Cont'd.) NET D I A L L E L SCORES CROSS II DARKNESS,HIGH TEMPERATURE F E M A L E 8. 949 2.449 5.699 2 1.250 2.277 1.763 3 1.450 11.700 6.575 4 0.799 3.450 2.125 5 5.500 3.750 4.625 6 2.090 5.000 3.545 7 2.699 5.450 4.075 10.100 4.650 7.375 1.350 4.300 2. 325 1.250 1.349 1.300 3.600 2.600 3.100 3.299 7.899 5.599 0.000 0.000 0.000 2.349 7.050 4.699 M A L E 6. 857 12.000 9.428 3.650 8. 000 5.825 6.099 0.000 3.050 6.100 6.684 6.392 3.000 3. 500 3.250 3. 849 9.450 6.649 0.000 1 .333 0.666 2.649 10.250 6.450 3.450 10.500 6.975 1.650 12.850 7.250 0.000 0.000 0.000 4.800 0.000 2.400 0.000 11.166 5.583 4. 800 3.850 4. 325 1. 599 6.350 3.974 3. 149 5.150 4.150 5.500 9.700 7.600 5.200 3.700 4.450 5.349 5.849 5 .599 0.600 6.000 3.300 6.099 5.849 5.974 16.000 6.666 11.333 11.750 1.800 6.775 9.250 18.250 13.750 13.500 3.000 8.250 6.052 7.899 6.976 5.000 5.500 5.250 5.600 4. 500 5.050 5. 700 8. 899 7.300 5. 050 1.750 3.400 5.650 5.350 5.500 4.900 14.800 9.850 8 .649 6.800 7.724 7.875 10.285 9.080 8.650 6.349 7.500 62 Appendix 2 (Cont'd.) NET DIALLEL SCORES CROSS I DAYLIGHT,MEDIUM TEMPERATURE F E M A L E 1 2 3 4 . 5 6 7 14.600 20.150 17.700 7.450 21.650 22.312 10.850 22.000 17.700 11.900 18.900 22.500 23.875 18.350 18.300 18.925 14.800 13.175 22.075 23.093 14.600 14.550 15.250 16.450 19.210 16.850 20.333 20.050 19.400 20.600 21.600 13.899 17.700 23.111 22.800 16.975 17.925 19.025 16.555 17.275 21.722 21.425 3 21.600 17.650 22.400 19.150 16.850 20.400 19.000 16.200 22.800 22.700 16.500 20.500 19.600 22.550 M 18.900 20.225 22.550 17.825 18.675 20.000 20.775 A 4 13.600 18.400 20.000 15.950 23.350 20.538 16.750 L 21.950 16.550 15.400 23.800 21.600 10.000 18.650 E 17.775 17.475 17.700 19.875 22.475 15.269 17.700 17.650 19.500 17.450 24.000 20.050 21.222 22.800 16.150 16.050 19.600 17.800 16.750 20.450 20.300 16.900 17.775 18.525 20.900 18.400 20.836 21.550 16.950 17.150 22.050 14.400 18.350 21.631 21.350 10.050 21.000 20.400 14.050 20.750 21.600 17.600 13.500 19.075 21.225 14.225 19.550 21.615 19.475 7 17.800 15.200 18.600 15.000 20.850 17.647 18.650 17.750 21.050 22.300 19.550 16.950 21.846 18.894 17.775 18.125 63 Appendix 2 (Cont'd.) NET O I A L L E L SCORES CROSS II DAYLIGHT,MEDIUM TEMPERATURE F E M A L E 1 2 3 4 5 6 7 17.450 13.150 14.300 12.850 16.300 24.000 17.800 9.650 19.375 16.550 18.571 21.600 24.000 17.950 13.550 16.262 15.425 15.710 18.950 24.000 17.875 22.950 20.550 21.050 13.250 21.400 24.000 19.250 17.500 17.600 14.750 20.900 19.450 24.000 8.000 20.225 19.075 17.900 17.075 20.425 24.000 13.625 3 24.000 20.000 17.800 21.600 18.285 24.000 21.600 14.750 2^.000 24.000 21.000 12.000 24.000 24.000 M 19.375 22.000 20.900 21.300 15.142 24.OQ0 22.800 A 4 17.500 14.350 20.400 17.550 7.899 24.000 19.300 L 19.300 13.350 15.250 15.700 18.750 12.000 18.100 6 18.650 14.100 17.825 16.625 13.325 18.000 18.700 13.400 21.650 21.500 19.250 21.600 24.000 16.950 18.550 IS.400 16.500 17.300 20.650 18.000 23.100 15.975 19.525 19.000 18.275 21.125 21.000 20.025 23. 875 23.200 24. 000 22 .285 20.250 17. r'00 20.050 17.571 24.000 21.000 9.000 22.300 0.000 15.833 20.723 23.600 22.500 15.642 21.275 8.750 17.941 20.700 19.850 19.350 11.699 17.650 16.833 22.400 16.700 20.450 21.450 20.400 18.900 16.571 17.650 18.700 20.150 20.400 16.049 18.275 16.702 20.025 64 Appendix 2 (Cont'd.) NET DIALLEL SCORES CROSS I DARKNESStMEDIUM 'TEMPERATURE F E M A L E 1 2.250 3.350 2.800 2 0.000 2.650 1.325 3 5.850 4.800 5.325 4 8.437 8.550 8 .493 5 4.250 3.849 4.050 6 9.687 8.375 9.031 7 5.750 3.750 4.750 2.450 2. 349 2.400 5.000 6.100 5.550 3. 750 3.500 3.625 2.450 4.800 3.625 1.349 3.299 2.324 9.600 5.222 7.411 5.849 2. 399 4. 124 4. 300 2.649 2.899 2.399 2.399 6.999 4.250 6.600 6. 149 8.249 3.950 3.700 3.149 9.100 M A L E 3.474 6. 750 9.950 8.350 2.649 2.650 6.550 4.600 4.699 9.350 2.250 5.800 5.425 0.050 1.200 0.625 7. 199 4.550 3.650 4.100 3.825 5.384 11.470 8.427 6. 125 8.899 5.200 7.050 5.200 4.050 4.625 1 .700 5.399 3.550 3.900 2. 899 3.400 9.450 3.850 6.650 5.550 7.550 6.550 5.000 5.750 5.375 5.700 10.789 8.244 4.099 3.450 3.775 1.500 5.250 3. 375 0.400 5. 100 2.750 0.350 7. 150 3.750 8 .550 7.700 8.125 6.578 10.050 8.314 11.100 8. 100 9.600 3.250 10.750 3. 399 4.950 3.299 8.699 1.899 8.950 9.550 9.300 1.235 7.692 4.400 6.099 7.000 4.175 5.999 5.425 9.425 4.463 5.250 65 Appendix 2 (Cont'd.) NET DIALLEL SCORES CROSS II DARKNESS,MEDIUM TEMPERATURE F E M A L E 9. 300 12.750 11.025 2 4. 800 3.375 4.087 3 5.949 6.600 6.275 4 1.500 16.928 9.214 5 1. 150 9.350 5.250 6 10.000 12.000 11.000 7 5. 500 9.857 7.678 6. 500 6.157 6.328 0.050 8. 999 4.5?4 6.250 2.450 4.350 3.450 3.700 3.575 6.550 5 .950 6.250 24.000 24.000 24.000 8. 200 2.950 5. 575 M A L E 16.200 8. 250 12.225 14.800 7. 800 11.300 6.000 0.000 3. 000 5. 800 13.650 9.725 6.550 2.000 4. 275 7.250 2.450 4.850 5.099 3.000 4.050 5.000 10.200 7.600 7.461 10.500 8.980 2.549 7.349 4.949 12.000 12.000 12.000 0. 333 12.000 6.1 66 1. 700 2. 799 2.250 2. 150 7.200 4.675 1.900 10.249 6. 074 3. 399 3.578 3.489 4.050 7. 149 5.600 7.750 7.599 7.675 3.950 4.899 4.425 13.500 6.250 9.875 2.099 12.850 7.474 12.625 0.000 6.312 15.200 0. 000 7.600 23.500 20.000 21.750 17.142 15.000 16.071 10.500 4.399 7.450 10.500 24.000 17.250 8.950 0.000 4.475 8. 350 8.050 8.200 2. 599 3.050 2.825 5. 349 7.500 6.425 5 .650 10. 100 7.875 7.349 10.650 9.000 14.166 9. 142 11.654 4.000 7.599 5.800 66 Appendix 2 (Cont'd.) NET DIALLEL SCORES CROSS I DAYLIGHT,LOW TEMPERATURE F E M A L E 1 2 3 4 5 6 7 23.850 18.500 20.750 14.100 24.000 19.058 23.950 21.600 22.450 11.500 19.300 16.500 16.625 19.200 22.725 20.475 16.125 16.700 20.250 17.841 21.575 19.350 17.950 21.900 19.100 18.950 8.600 18.800 20.300 22.150 21.157 18.450 16.800 20.800 16.950 19.825 20.050 21.528 18.775 17.875 14.700 17,875 3 22.050 10.400 19.850 18.450 19.350 15.650 23.250 15.100 21.500 13.000 20.250 19.000 18.250 22.600 M 18.575 15.950 16.425 19.350 19.175 16.950 22.925 A 4 20.100 19.600 20.250 20.400 21.600 17.700 18.750 L 20.200 21.600 19.250 20.450 21.800 12.857 20.400 E 20.150 20.600 19.750 20.425 21.700 15.278 19.575 18.600 16.300 19.500 19.250 20.100 21.705 21.550 16.550 18.800 22.800 18.450 20.400 21.800 23.350 17.575 17.550 21.150 18.850 20.250 21.752 22.450 20.000 22.100 21.600 19.250 10.600 21.210 17.850 21.250 19.250 21.450 19.750 22.900 6.950 15.600 20.625 20.675 21.525 19.500 16.750 14.080 16.725 15.100 14.350 21.400 21.950 24.000 23.700 21.600 20.550 21.000 23.300 22.600 21.650 20.307 19.700 17.825 17.675 22.350 22.275 22.825 21.753 20.650 67 Appendix 2 (Cont'd.) NET DIALLEL SCORES CROSS II DAYLIGHT,LOW TEMPERATURE F E M A L E 1 2 3 4 5 6 7 12.500 15.157 15.450 13.200 19.200 20.000 18.150 17.900 17.533 17.722 15.666 16.800 24.000 19.200 15.200 16.345 16.586 14.433 18.000 22.000 18.675 21.800 20.900 20.350 21.600 13.050 15.500 18.600 24.000 15.000 17.150 19.850 16.750 15.500 13.899 22.900 17.950 18.750 20.725 17.400 15.500 16.250 3 21.25C 13.000 18.950 19.500 19.200 11.500 16.800 18.000 16.000 12.000, 12.000 0.000 11.500 20.000 M 19.625 14.500 15.475 15.750 9.600 11.500 18.400 A 4 18.750 15.850 17.800 12.750 20.400 0.000 18.250 L 16.400 9.900 19.250 17.950 17.800 11.666 20.000 E 17.575 12.875 18.525 15.350 19.100 5.833 19.125 18.400 15.350 19.700 15.500 15.800 17.000 20.500 23.900 15.200 21.850 16.400 18.850 12.000 17.450 21.150 15.275 20.775 15.950 17.325 14.500 18.975 17. 500 19.200 0.000 14.846 19.450 16.r»00 13.210 15.000 3.000 0.000 21.000 15.300 16. n00 10.400 16.250 11.100 0.000 17.923 17.375 16.OQ0 11.805 19.950 10.750 21.950 20.300 16.200 20.000 17.900 15.400 17.650 16.450 16.750 13.800 20.571 18.600 17.675 14.200 19.200 18.525 15.000 20.285 18.250 6 8 Appendix 2 (Cont'd.) NET DIALLEL SCORES CROSS I DARKNESS»LOW TEMPERATURE F E M A L E 1 10.450 11.100 10.775 2 6. OOO 8.200 7. 100 3 10.100 5.849 7.974 4 4.800 14.400 9.600 5 7. 149 9.050 8.100 10.764 11.888 11.326 7 5. 736 7.450 6. 593 9. 149 7. 300 8.225 4.050 4.050 4.050 6.500 5.099 5.800 8.750 7.700 8.225 9.050 3.900 6.475 3.199 3.466 3.333 9.050 6.800 7.925 5.050 5. 199 4.999 6.050 5.750 6. 199 3.7 99 3 .949 6. 300 7.500 10.450 7.350 6.099 7. 300 M A L E 5.125 13.350 8.750 11.050 5.525 6.750 13.050 9.900 5.975 7.949 13.750 10.850 3.874 6.200 9 .449 7.325 6.900 9. 149 11.000 10.075 8.900 4.800 3.214 4.007 6.699 11.550 9.800 10.675 10.149 3. 900 7.024 8.500 11.250 9.375 8.399 10. 150 9.274 6.599 4. 300 5.449 8.500 6.300 7.400 7.000 6.000 6.500 6. 800 7.950 7. 375 6.100 4. 500 7.000 6.650 9.600 9. 150 7.399 8.200 4.900 7.000 10.894 4.700 5.950 5.300 5.300 6.825 9.375 7.800 5 .950 7.797 5.625 9.950 12.850 10.000 7.349 8.450 6. 900 7.400 11.500 7.300 10.950 14.266 15.461 3. 150 11.050 11.400 8.675 7.675 9.450 9.125 1 4 . 3 6 4 7. 100 69 Appendix 2 (Cont'd.) NET DIALLEL SCORES CROSS I I DARKNESS,LOW TEMPERATURE F E M A L E 15.650 16.789 2 4.166 8.066 12.750 7.777 4 13.100 9.000 5 7.450 9.900 6 16.000 O.riOO 7 1.349 3. 299 16.219 6.116 10.263 11.050 8.675 8.000 2.324 7. 599 12.850 6.249 1 .850 8.500 4.950 12.700 10.450 10.149 6.650 24.000 24.000 5. 550 7.750 10.225 4.050 6.725 11.575 8.399 24.000 6.650 M A L E 20.750 7.250 14.000 12.050 7.050 9. 550 11.666 0.000 5.833 13.150 8.950 11.050 11.050 5.500 8.275 18.700 14.350 16.525 6.399 1.285 3.842 15.700 13.450 14.575 13.100 11.000 12.050 12.450 14.650 13.550 0.^00 0.000 0.^00 12.000 10.000 11.000 3. 200 1. 800 2.500 15.200 9. 100 13.650 4. 599 9.600 7.099 6.050 6.250 6.150 10.250 11.300 10.775 6.550 12.550 9.550 5 .450 7.199 6.325 5.000 8.750 6.875 3.699 5.649 4.674 11.375 6.285 8. 830 9.200 0. 500 4.850 0.000 12.000 6.000 5.538 10.125 7.831 5.500 7.050 6.275 4.000 4.000 4.000 1.736 6.400 4. 068 7. 099 13.400 10.250 2. 150 6.800 4.475 8.050 8.650 8.350 4.600 10.300 7.450 5.650 3.900 4.775 13.666 8.714 11.190 4.250 4.849 4.550 Appendix.3. Analysis of variance tables for the d i a l l e l a n a l y s i s using [layman's model for r e c i p r o c a l e f f e c t s . , (A) Tests run at the high temperature (17.5 + 1.5°C) for the net scores. Cross I Daylight Cross II Daylight Cross I Darkness Cross II Darkness Source d.f. M.S. F M.S. . F+ M.S. F+ M.S F+ a 6 27.6620 5.94' * 27.2949 1.18 n.s. 7.9520 0 .5514 n.s. 16.8775 1.28 n. s. b l 1 46.1886 3.72+ n. s. 31.5161 1.36 n.s. 35.1737 2 .4394 n.s. 0.4710 0.04 n. s. b2 6 8.5557 0.69+ n. s. 17.8158 0.77 n.s. 9.2285 0 .6400 n.s. 3.0008 0.23 n. s. b3 14 8.1307 0.65+ n. s. 21.7497 0.94 n.s. 10.5877 0 .7342 n.s. 7.7807 0.59 n. s. b 21 10.0644 0.81+ n. s. 21.0908 0.91 n.s. 11.3701 0 .7885 n.s. 6.0670 0.46 n.s. c 6 32.17*0 2.59+ 87 .0735 3.77 ** 18.1852 1 .2612 n.s. 52.2473 3.96 *•* d 15 15.4602 1.24+ n. s. 38.0432 1.65 n.s. 12.2430 0 .8490 n.s. 13.7047 1.03 n. s. Blocks 1 18.3208 1.48 n. s. 16.4132 0.71 n.s. 0.5622 0 .04 n.s. 40.8078 3.10 n. s. B X a 6 4.6601 16.9729 28.8467 18.9670 B X b l 1 8.8917 0.8962 3.7850 2.7880 B x b2 6 10.3562 27.7934 10.1448 17.4030 B X b3 14 15.5299 34.5087 9 .6880 10.6208 B X b 21 13.7356 30.9895 9.5374 12.1855 B X c 6 26.2056 10.9344 29.0964 9.9279 B X d 15 8.1692 19.4308 9.6107 13.5587 Blocks pooled 48 12.4204 23.1184 14.4189 13.1801 •"Each item tested against i t s own block i n t e r a c t i o n . + A l l items tested against the pooled i n t e r a c t i o n mean square. (cont'd') Appendix 3 (cont'd) (B) Test run at the medium temperature (9.7 + 0.5°C) for the net scores. Source d.f. Cross I Daylight Cross II Daylight Cross I Darkness Cross II Darkness M.S. F+ M.S. F+ M.S. F+ M.S. F a 6 19.1258 1.82 n.s. 25.6927 1.48 n.s. 20.5566 3.60 125.7276 6.16+ *** bl 1 13.3988 1.27 n.s. 34.4385 1.98 n.s. 2.8547 0.50 n.s. 0.0224 0.00+ n.s. b2 6 9.1491 0.87 n.s. 56.7009 3.26 -kit 17.3922 3.04 * 5.5384 0.27+ n.s. b3 14 10.6299 1.01 n.s. 15.2849 0.88 n.s. 6.7310 1.18 n.s. 33.9109 1.66+ n.s. b 21 10.3386 0.98 n.s. 28.0301 1.61 n.s. 9.5924 1.68 n.s. 24.1907 1.19+ n.s. c 6 7.6317 0.72 n.s. 5.7868 0.33 n.s. 3.0485 0.53 n.s. 19.7808 0.97+ n.s. d 15 10.6309 1.01 n.s. 11.6144 0.67 n.s. 8.0756 1.41 n.s. 38.1929 3.10* Blocks 1 10.8987 1.04 n.s. 46.0210 2.64. n.s. 35.4910 6.21 7.8356 0.38 n.s. B X a 6 12.0426 24.9864 2.5714 24.7583 B X bl 1 14.3980 33.3538 3.7362 61.3862 B X b2 6 7.0447 16.2524 1.5818 35.1984 B X b3 14 18.8288 20.2152 7.0573 14.0008 B X b 21 15.2509 19.7086 5.3347 22.3137 B X c 6 3.3569 16.0333 8.7578 29.5518 B X d 15 6.1749 11.6868 6.2915 12.3286 Blocks pooled 48 10.5269 17.4021 5.7162 20.4037 *Each item tested against its own block interaction. +A11 items tested against the pooled interaction mean square. (cont'd) Appendix 3 (cont'd) (C) Test run at the low temperature (5.0 + 1.5°C) f o r the net scores. Cross I Daylight Cross II Daylight Cross I Darkness Cross I I Darkness Source d.f. M.S. F+ M.S. F+ M.S. F M.S. F a 6 17.5911 1.47 n.s. 51.1315 3.63 13.8716 2.63 + 68.4220 4.70 + *** b l 1 0.2229 0.02 n.s. 0.3687 0.03 n.s. 4.7018 19.15* n.s. 0.5583 0.04 + n.s. bl 6 12.7076 1.07 n.s. 19.4571 1.38 n.s. 4.7119 0.99 n.s. 22.3197 8.94 b3 14 6.9123 0.58 n.s. 26.8248 1.90 7.9638 2.75 * 30.3344 2.08 + b 21 8.2496 0.69 n.s. 23.4599 1.67 n.s. 6.8793 2.09* 26.6266 1.83 + * c 6 4.0703 0.34 n.s. 30.6211 2.17 n.s. 17.6833 3.36 + ** 66.5623 4.57 + *** d 15 13.4789 1.13 n.s. 36.5324 2.59 11.6424 2.21 + * 28.3688 3.94 ** Blocks 1 0.0097 0.00 n.s. 20.6213 1.46 n.s. 2.2863 0.43 n.s. 21.6437 1.49 n.s. B X a 6 14.3370 15.7473 7.2967 18.1368 B X b l 1 36.2248 5.2299 0.2455 0.3785 B X b2 6 13.6220 5.7675 4.7460 2.4976 B X b3 14 10.4737 14.7840 2.8931 24.4343 B X b 21 12.5994 11.7529 3.2964 17.0212 B X c 6 5.7127 16.1852 7.0127 20.8001 B X d 15 12.5088 15.8438 6.5089 7.2007 Blocks pooled 48 11.9275 14.0846 5.2649 14.5641 *Each item t e s t e d against i t s own block i n t e r a c t i o n . +A11 items t e s t e d against the pooled i n t e r a c t i o n mean square. (cont'd) Appendix 3 (cont'd) (D) Crosses I and I I summed f o r the net scores - h i g h temperature (17.5 + 1.5°C). D a y l i g h t Darkness Source d.f. M.S. F+ M.S. F+ a 6 56.3204 1.62 n.s. 30.5441 1.59 n. s. b l 1 154.0260 4.42 * 32.0418 1.67 n. s. b2 6 10.9949 0.32 n.s. 12.1986 0.63 n. s. b3 14 28.8923 0.83 n.s. 15.7424 0.82 n. s. b 21 29.7375 0.85 n.s. 15.5060 0.81 n. s. c 6 146.6446 4.21 ** 80.1238 4.17 ** d 15 55.8417 1.60 n.s. 36.1977 1.88 * Bl o c k s 1 69.4127 1.99 n.s. 37.4285 1.94 n. s. B X a 6 29.1281 28.6805 B x b l 1 15.4347 0.4788 B X b2 6 28.8990 15.0303 B X b3 14 53.8079 17.7032 B X b 21 44.8638 16.1193 B X c 6 25.3117 35.8121 B X d 15 26.9524 13.1860 Bloc k s pooled 48 34.8555 19.2344 *Each item t e s t e d a g a i n s t i t s own b l o c k i n t e r a c t i o n . +A11 items t e s t e d a g a i n s t the pooled i n t e r a c t i o n mean square. (cont'd) Appendix 3 (cont'd) (E) Crosses I and I I summed f o r the net sco r e s - medium temperature (9.7 + 0.5°C). D a y l i g h t Darkness Source d.f . M.S. F+ M.S. F+ a 6 82.6032 2.56 * 187.1608 6.48 *** b l 1 4.9665 0.15 n.s. 2.3457 0.08 n.s. b2 6 50.3476 1.56 n.s. 13.8296 0.48 n.s. b3 14 33.5124 1.03 n.s. 45.4321 1.57 n.s. b 21 36.9631 1.15 n.s. 34.3511 1.19 n.s. c 6 16.6310 0.52 n.s. 24.7878 0.86 n.s. d 15 27.7803 0.86 n.s. 45.8894 1.59 n.s. B l o c k s 1 12.1264 0.38 76.6764 2.65 n.s. B x a 6 46.3912 23.1447 B X b l 1 3.8107 95.3973 B X b2 6 19.9306 34.6888 B x b3 14 48.1729 35.2081 B X b 21 37.9912 37.9259 B X c 6 30.8300 28.4132 B X d 15 19.0824 18.7283 B l o c k s pooled 48 32.2370 28.8899 *Each i t e m t e s t e d a g a i n s t i t s own b l o c k i n t e r a c t i o n . +A11 items t e s t e d a g a i n s t the pooled i n t e r a c t i o n mean square. (cont'd) Appendix 3 (cont'd) (F) Crosses I and II summed f o r the net scores - low temperature (5.0 + 1.5°C). Daylight Darkness Source d.f. M.S. F+ M.S. F+ a 6 129.9008 5.50 *** 99.5462 5.28 *** 1 0.3468 0.01 n.s. 8.2620 0.44 n.s. bl 6 11.3712 0.48 n.s. 36.3138 1.92 n.s. b3 14 42.6111 1.81 n.s. 30.8613 1.64 n.s. b 21 31.6728 1.34 n.s. 31.3430 1.66 n.s. c 6 38.2083 1.62 n.s. 119.7470 6.35 *** d 15 53.2132 2.25 * 42.8404 2.27 * Blocks 1 12.4879 0.53 n.s. 10.5000 0.56 n.s. B X a 6 20.4531 27.5536 B X b l 1 16.8327 0.0063 B X b2 6 30.8994 5.6309 B X b3 14 13.1881 23.5509 B X b 21 18.4220 17.3097 B X c 6 14.9680 23.5292 B X d 15 35.5740 15.7111 Blocks pooled 48 23.6041 18.8681 'Each item t e s t e d a g a i n s t i t s own block i n t e r a c t i o n . + A l l items t e s t e d a g a i n s t the pooled i n t e r a c t i o n mean square. 

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