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Behavioural ecology of the longnose dace, Rhinishthys cataractae (Pisces:Cyprinidae) : significance of… Bartnik, Victor George 1973

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<7 BEHAVIOURAL ECOLOGY OF THE LONGNOSE DACE, RHINICHTHYS CATARACTAE (PISCES, CYPRINIDAE): SIGNIFICANCE OF DACE SOCIAL ORGANIZATION by VICTOR GEORGE BARTNIK B.Sc. (Hons.), Uni v e r s i t y of Manitoba, 1968 M.Sc, Un i v e r s i t y of Manitoba, 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY ,in the Department of ZOOLOGY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1973 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of 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 of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of ZOOLOGY  The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8 , Canada Date i i ABSTRACT The s o c i a l behaviour of a stream population of longnose dace, Rhinichthys cataractae d u l c i s , i s described. Both males and females occupy the same r i f f l e areas and defend t e r r i t o r i e s (approx. 10 cm i n diameter) during the breeding phase. In addition, male dace are evidently attracted to and i n t e r a c t a g o n i s t i c a l l y with other males r e s u l t i n g i n the formation of c l u s t e r s of male t e r r i t o r i e s . During the night, females leave t h e i r t e r r i t o r i e s to court and spawn with t e r r i -t o r i a l males. A f t e r spawning, males show strong nest s i t e attachment and are i n h i b i t e d from eating eggs. Parental males remain d i r e c t l y over the nest s i t e and frequently probe the substrate with t h e i r snouts. They defend the area against a l l f i s h with the exception of receptive females with which they may spawn. Comparison with a stream population of the sub-species, R. cataractae  cataractae, reveals differences i n phenotypic appearance, d i u r n a l rhythm of breeding a c t i v i t y , and female t e r r i t o r i a l i t y . Unlike R.c.. d u l c i s , R.c^. cataractae are reproductively active during daylight hours and males dis p l a y bright n u p t i a l c o l o r a t i o n . Breeding c o l o r a t i o n i n males of R.£. d u l c i s i s f a i n t or absent. Unlike R.c. d u l c i s , males and females of R.c. cataractae are segregated i n t o d i f f e r e n t habitats and females remain n o n - t e r r i t o r i a l . Experimental analyses of the p h y s i c a l and b i o t i c f a c t o r s involved i n the causation of t e r r i t o r i a l behaviour were conducted i n a laboratory stream tank. The major p h y s i c a l f a c t o r involved i n the s e l e c t i o n of t e r r i t o r i e s by male dace i s the presence of coarse gravel s u i t a b l e f o r i i i spawning. Female dace s e l e c t and defend only enclosed areas which provide both overhead cover and s h e l t e r from current. Females prefer shelters which contain food and thus the presence of food may be a proximate f a c t o r involved i n the s e l e c t i o n of t e r r i t o r i e s by female dace. Laboratory tests i n d i c a t e that female t e r r i t o r i a l behaviour i s e l i c i t e d by i n t e r a c t i o n with breeding males. Outside the breeding phase, when t e r r i t o r i a l i t y i s relaxed, male and female dace d i f f e r l i t t l e i n the areas they choose to occupy. The experimental evidence suggests that the functions of male t e r r i t o r y are: i ) provision of space i n which males can court and spawn with females with minimal interference from other males, and i i ) p r otection of eggs from i n t r a s p e c i f i c predation. For females, t e r r i t o r i a l i t y apparently functions to reduce c o n f l i c t s with males ( i . e . , reduce court-ship harassment and attacks from males). Changes i n the behaviour of males occur a f t e r spawning and coincide with periods of greatest egg v u l n e r a b i l i t y . These post spawning behavioural a c t i v i t i e s of males protect f r e s h l y deposited eggs while making them les s accessible to predators. The evidence also suggests that a major function of t e r r i t o r y i n both sexes i s the p r o v i s i o n of s h e l t e r from current. Dace not defending a she l t e r may be forced to make frequent movements i n the strong current of the r i f f l e habitat. Dace swimming against strong currents for even short periods ( i . e . , 5 min) become fatigued and lose t h e i r a b i l i t y f o r co-ordinated locomotion. Such stressed i n d i v i d u a l s may be vulnerable to predation. The data also suggest that dace t e r r i t o r i a l behaviour may act as a dispersing mechanism, thereby l i m i t i n g the density of breeding f i s h i n a l o c a l i z e d area. i v T e r r i t o r i a l males of clustered groupings remain behaviourally synchronized. They display strong s i t e attachment and i n t e r f e r e l i t t l e with the reproductive a c t i v i t i e s of neighbouring males. Consequently, these s o c i a l groupings function to further reduce interference from c o n s p e c i f i c s . Assemblages of more widely spaced t e r r i t o r i a l males, created i n the laboratory, experienced greater i n t r a s p e c i f i c interference and egg predation than did males of clustered t e r r i t o r i a l groupings. Thus i t seems most probable that i n t r a s p e c i f i c interference and egg predation have provided a major s e l e c t i o n pressure favouring both male t e r r i t o r i -a l i t y and t e r r i t o r y c l u s t e r i n g i n longnose dace. :.v TABLE OF CONTENTS Page LIST OF TABLES x i LIST OF FIGURES . .... x i i i ACKNOWLEDGEMENTS x v l i INTRODUCTION 1 MATERIALS AND METHODS . 4 A. Study Area 4 B. Holding Conditions ^ C. Description of Stream Tank 6 D. Experimental Channels 6 E. Experimental Conditions 8 F. Experimental F i s h 9 SECTION I: THE SOCIAL BEHAVIOUR OF LONGNOSE DACE • H A. Description of the Behaviour of R.c_. d u l c i s H a. Reproductive Behaviour H b. Agonistic Behaviour Patterns Recorded During 1* Observations c. Courtship Behaviour Patterns Recorded During Observations ; ^ d. Egg Eating I n h i b i t i o n ^ i . Outline of Experiment ^ i i . Procedure 17 i i i . Experimental/Fish 18 i v . Results and Discussion 18 e. Comparison with R. £. cataractae 20 Page B. Summary and Discussion . 27 SECTION I I : FACTORS INVOLVED IN THE CAUSATION OF TERRITORIAL BEHAVIOUR AND THE SELECTION OF A TERRITORY 31 A. Breeding Phase 32 a. Experimental Analysis of the Factors Involved i n the Selection of T e r r i t o r i e s by Male and Female Dace 32 i . Outline of Experiment ;. 32 i i ; Procedure. 35 i i i . Experimental F i s h . . . . . . 37 i v . Results and Discussion 37 b. S o c i a l Factors A f f e c t i n g T e r r i t o r i a l Behaviour i n Female R. c_._ d u l c i s 45 i . Outline of Experiment 45 i i . Procedure........ 45 i i i . Experimental F i s h . 46 i v . Results and Discussion 46 c. S o c i a l Factors A f f e c t i n g T e r r i t o r i a l Behaviour i n Female R.c:. cataractae .................. 49 i . Outline of Experiment.... 49 i i . Procedure 49 i i i . Experimental F i s h 51 i v . Results and Discussion 51 d. S o c i a l Factors A f f e c t i n g T e r r i t o r i a l Behaviour i n Male R.c. d u l c i s 56 via. Page e. Clustering of Male T e r r i t o r i e s . . . 58 i . Outline of Experiment 58 i i . Procedure . ... ,. 60 i i i . Experimental Fi s h 62 i v . Results and Discussion .. ... 62 f. Behaviour of Clustered T e r r i t o r i a l Male Dace... 66 i . Outline of Experiment 66 i i . Procedure 67 i i i . Experimental Fi s h • ^ i v . Results and Discussion ... 69 g. The Role of Food i n the D i s t r i b u t i o n of Dace T e r r i t o r i e s 73 i . Outline of Experiment... 73 i i . Procedure 73 i i i . Experimental F i s h 75 i v . Results and Discussion , 75 B. Non-Breeding Phase .... 80 a. S i t e Preferences of Male and Female Dace i n Late Summer and Winter.. . ... . 80 i . ' Outline of Experiment 80 i i . Procedure 82 i i i . Experimental Fi s h 82 i v . Results and Discussion .... 82 v i i i Page b. Seasonal V a r i a t i o n i n the Spacing Patterns of Dace 88 i . Outline of Experiment........ 88 i i . Procedure 88 89 i i i . Experimental F i s h i v . Results and Discussion 89 c. Role of Food i n the D i s t r i b u t i o n of Dace i n LateSSummer 93 i . Outline of Experiment 93 i i . Procedure .... 93 i i i . Experimental F i s h 93 i v . Results and Discussion • 94 C. Spacing Patterns of Dace Fry/ 94 i . Outline of Experiment 94 i i . Procedure 97 i i i . Results and Discussion........ 97 D. Discussion of the Causation of T e r r i t o r i a l Behaviour 99 SECTION I I I : THE FUNCTIONAL SIGNIFICANCE OF THE SOCIAL ORGANIZATION OF DACE.. 104 A. Introduction 104 B. Inferences from Observations and Experiments i n Section II as to the Function of Various Aspects of T e r r i t o r i a l Behaviour 105 i x Page C. Experimental Tests of Hypotheses Regarding Function.... 106 a. S i g n i f i c a n c e of T e r r i t o r y Clusters 106 i . Outline of Experiment.. .. 106 i i . Procedure.....:. ••• 107 i i i . Experimental F i s h •••• 109 i v . Results and Discussion 110 b. S i g n i f i c a n c e of Post Spawning Changes i n Male Behaviour 118 c. S i g n i f i c a n c e of Male T e r r i t o r y as Determined by V u l n e r a b i l i t y of Dace Eggs to I n t r a s p e c i f i c Predation .. . 122 i . Outline of Experiment ...... 122 i i . Procedure............ 122 i i i . Experimental F i s h 124 i v . Results and Discussion 125 d. Other Predators ... 129 i . Trout... ... 129 i i . Sculpins • • 130 i i i . Egg Predation... 131 e. S i g n i f i c a n c e of Female T e r r i t o r y as Determined by Changes i n Blood Lactate i n Dace a f t e r Exercise 134 i . Outline of Experiment 135 i i . Procedure . 136 i i i . Experimental F i s h 138 i v . Results and Discussion 139 X Page f. T e r r i t o r i a l Behaviour as a Dispersing Mechanism 141 i . Outline of Experiment 141 i i . Procedure..... •• 142 i i i . Experimental F i s h 144 i v . Results and Discussion... 144 D. Discussion of the Significance of S o c i a l Organization i n Dace • • • • 148 LITERATURE CITED • 154 X X LIST OF TABLES Table Page I. . Number of eggs eaten by d i f f e r e n t types of dace presented with recently f e r t i l i z e d eggs 19 I I . A g o n i s t i c behaviour patterns commonly performed by R.-c. cataractae and R._c. d u l c i s during the breeding phase.. 24 I I I . Environmental features incorporated into experimental structures.... 34 IV. Mean number of ago n i s t i c acts won per 30 min by male and female dace against the same and opposite sex during day and night • 39 V. T o t a l number of agonistic acts won by males against other males at coarse substrate structures during night 56 VI. Actual mean distance between four t e r r i t o r i a l male dace as compared with maximum and minimum mean distances attainable 63 VII. Actual mean distance between four t e r r i t o r i a l male dace as compared with maximum and minimum mean distances attainable before and a f t e r screen d i v i d e r was removed 63 VIII. Mean number of ago n i s t i c acts won per food and no food area by male and female dace against the same and opposite sex during day and night 78 IX. Mean number of ago n i s t i c acts won per area per 30 min by male and female dace during l a t e summer 83 X. Agonistic behaviour patterns commonly performed by breeding and non-breeding dace '. 84 x i i Table Page XI. Mean number of agonistic acts won and feeding acts performed by dace per food, no food, and open area during l a t e summer.... 95 XII. Number of substrate contacts, agonistic acts won, and eggs eaten by d i f f e r e n t types of dace at nests spawned i n c l o s e l y and widely spaced t e r r i t o r i a l groupings 112 XIII. Number of eggs eaten by dace i n Experiments A and B.... 117 XIV. Number of substrate contacts made, and eggs eaten by transient and parental dace at 0, 4, 24, and 72 hr o l d nests • 127 XV. Frequency of occurrence of eggs i n Cbttus asper. Cj. al e u t i c u s , and Salmo gairdheri stomachs when only Rhinichthys eggs (1971) or Mylocheilus eggs (1972) were abundant i n r i f f l e s from where sculpins and trout were c o l l e c t e d 133 XVI. Frequency of occurrence of eggs i n Rhinichthys  cataractae stomachs when Rhinichthys eggs were abundant i n r i f f l e s from where dace were c o l l e c t e d (1971) 133 XVII. Blood l a c t a t e values f o r (A) Non-exercised and (B) Exercised dace 140 x i i i LIST OF FIGURES Figure Page 1. South Alouette River and locations of s t a t i o n s . ... 5 2A. Top view of the stream tank 7 2B. Observational channel of the stream tank 7 3. D i e l p e r i o d i c i t y of agonistic and courtship a c t i v i t y of R._c. d u l c i s 12 4. Time i n t e r v a l s over which dace spawnings were observed or occurred but were not observed • 13 5. Rhinichthys cataractae cataractae and R.c d u l c i s males from Mink River, Manitoba, and Alouette River, B r i t i s h Columbia . .. 22 6. D i s t r i b u t i o n of adult dace i n rock r i f f l e s during pre-breeding and breeding periods •.. 26 7. Diagramatic sketches of s i x combinations of structures and bottom substrates placed i n the experimental channel. 33 8. Mean number of agonistic acts won per area per 30 min by breeding male and female dace during day and night.... 38 9. T o t a l number of a g o n i s t i c acts won and l o s t by i n d i v i d u a l male and female dace at d i f f e r e n t areas i n Replicates I, I I , and I I I ..... 42 10. Mean number of courtship acts performed per area per 30 min by male and female dace during day and night 44 11. Percent occurrence of d i f f e r e n t group sizes i n Channel 1 and Channel 2 . . 47 x i v Figure Page 12. T o t a l number of agonistic acts won by R.c^. d u l c i s females i n Channel 1 and Channel 2 ...... 47 13. Number of ago n i s t i c acts won by male and female R.J;. cataractae against the same and opposite sex during 3 days of 'forced' i n t e r a c t i o n and a fourth day not forced , 52 14. Frequency of occurrence of ag o n i s t i c behaviour patterns performed by R . c a t a r a c t a e females during 3 days of 'forced' i n t e r a c t i o n with males 54 15. Pattern of a g o n i s t i c and courtship a c t i v i t y performed by dace during the 72 hr before spawning at preferred structures 57 16. T y p i c a l sequence of ag o n i s t i c and courtship a c t i v i t y performed at a preferred structure 59 17A. Arrangement of structures i n channel (top view)... 61 17B. Pattern of ag o n i s t i c a c t i v i t y performed by t e r r i t o r i a l males during eight successive night observation periods 61 18. Frequency of substrate probing and time spent on nest by parental males with and without i n t e r a c t i o n with conspecific males • 7u 19. Latency to attack by parental males for each of f i v e successive i n t e r a c t i o n s with i n t r u d i n g conspecific males ^2 X V . Figure Page 20. Mean number of ag o n i s t i c , courtship, and feeding acts performed per food and no food area by male and female dace during day and night 76 21. Percent occurrence and t o t a l number of agonistic acts won at food, no food, and open areas by male dace during day and night...... 81 22. - Percent occurrence of male and female dace at d i f f e r e n t areas during l a t e summer.... ... 85 23. Percent occurrence of male and female dace at d i f f e r e n t areas during winter 87 24. Percent occurrence of d i f f e r e n t group si z e s of dace during spring, l a t e summer, and winter seasons 90 25A. Relationship between t o t a l number of agonistic acts performed and mean group sizes of dace during spring, l a t e summer, and winter seasons..... 91 25B. Relationship between t o t a l number of agonistic acts performed, number of ago n i s t i c acts won, and d i f f e r e n t group sizes of dace during spring, l a t e summer, and winter seasons............ 91 26. Mean percent occurrence of male and female dace per food, no food, and open area during l a t e summer 96 27. Arrangement of BC structures i n channel to induce (A) c l o s e l y spaced or clustered male t e r r i t o r i e s and (B) more widely spaced male t e r r i t o r i e s 108 x v i . Figure Page 28. Mean number of substrate contacts, agonistic acts won against transient males, and eggs eaten by d i f f e r e n t types of male dace at nests spawned i n clustered and widely spaced t e r r i t o r i a l groupings....... m 29A. Total number of agonistic acts won by male and female dace i n experiments A and B before and a f t e r spawning... 115 29B. Levels of a g o n i s t i c a c t i v i t y performed at the nest s i t e i n experiments A and B before and a f t e r spawning... 115 30. Agonistic a c t i v i t y performed by a t e r r i t o r i a l male before and a f t e r spawning .... 120 31. Number of eggs eaten and substrate contacts made by transient males at 0, 4, 24, and 72 hr old nests........ 126 32. Diagram of swim tunnel used to exercise dace............ 137 33A. Diagram of experimental channel i l l u s t r a t i n g r i f f l e and pool zones 143 33B. Group s i z e s and d i s t r i b u t i o n of dace at increasing de n s i t i e s during breeding and non-breeding phases....... 143 34. Relationship, between percent occurrence of dace i n pool and mean number of agonistic acts won per 30 min at increasing d e n s i t i e s during breeding and non-breeding phases ......... 146 r x v i i . ACKNOWLEDGEMENTS I thank Dr. N.R. L i l e y , my supervisor, for h i s encouragement during the study and his many h e l p f u l c r i t i c i s m s of the manuscript. I am most g r a t e f u l to Dr. J . M. Cullen f or h i s stimulating discussions with me. Drs. J.R. Krebs, T.G. Northcote,and J.D. McPhail also provided valuable suggestions and guidance. I am indebted to a l l the following people who helped me p u l l a seine: N. Stacey, B. Wishlow, J . MacLean, J . McClurg,and E. Kliewer. Blood l a c t a t e analyses were done with the help of Mr. R. Stanley. F i n a l l y I wish to thank my wife, Gwen, whose assistance both i n the f i e l d and lab made t h i s study pos s i b l e . F i n a n c i a l support for both the research and the author was provided through a Fi s h e r i e s Research Board Block Grant. 1 INTRODUCTION Fishes show a l l types of dispersion from b a s i c a l l y s o l i t a r y to highly organized schooling, and further, the dispersion frequently changes from one type to another during development. Seasonal cycles modify s o c i a l behaviour markedly. The seasonal temperature change and the sexual cycle cause perhaps the most prominent differences i n the attitudes of i n d i v i d u a l s . Although s o c i a l organizations have been studied i n t e n s i v e l y i n a v a r i e t y of f i s h species, attention has usually been given to des-c r i p t i v e aspects alone. Such studies give r i s e to i n t e r e s t i n g questions concerning the b i o l o g i c a l s i g n i f i c a n c e of these s o c i a l responses i n nature. Answers are frequently suggested but r a r e l y accompanied by objective evidence. A notable exception i s the recent study of the s i g n i f i c a n c e of t e r r i t o r i a l i t y i n three-spined sticklebacks conducted by van den Assem (1967). The longnose dace, Rhinichthys cataractae (Valenciennes), has the widest d i s t r i b u t i o n of any North American c y p r i n i d . I t occurs from coast to coast i n Canada, as f a r south as the Rocky Mountains i n Mexico, and as f a r north as the Mackenzie River near the A r c t i c C i r c l e (Carl et a l . , 1959). Longnose dace are found i n s w i f t l y flowing streams and occasionally i n lakes. E c o l o g i c a l studies conducted by Becker (MS, 1962) , Gee and Northcote (1963) , and Bartnik (1970) have revealed that the adults of t h i s c y p r i n i d occur mainly i n spaces between stones i n the rapids areas of streams where they feed p r i m a r i l y on bottom i n s e c t s . 2 As a consequence of a strong preference f o r fast water h a b i t a t s , longnose e x h i b i t highly contagious d i s t r i b u t i o n s within streams. The terete shape and negative buoyancy of older dace are adaptations to l i f e i n swift flowing waters. Newly emerged longnose are n e u t r a l l y buoyant and aggregations occupy shallow s t i l l waters along shores. Later i n t h e i r f i r s t year, these f i s h move into areas of higher water v e l o c i t i e s (Gee, 1968). Carl e_t a l . , (1959) state that the adult longnose dace i s apparently a s o l i t a r y rather than a schooling f i s h . However, with exception of recent studies of breeding habits (Bartnik, 1970, 1972), there i s v i r t u a l l y nothing i n the l i t e r a t u r e on the behaviour of t h i s ubiquitous species. A substantial population of Khinichthys cataractae  d u l c i s near the Univ e r s i t y of B r i t i s h Columbia at Vancouver made t h i s species s u i t a b l e for experimental i n v e s t i g a t i o n of several aspects of s o c i a l behaviour. The aims of t h i s study were: 1) to provide an accurate d e s c r i p t i o n of dace s o c i a l behaviour, 2) to determine the causal factors involved i n the p a r t i c u l a r s o c i a l organization observed, and 3) to present ob-j e c t i v e evidence as to the fun c t i o n a l s i g n i f i c a n c e of dace s o c i a l organi-zation. Dace were studied during both breeding and non-breeding phases of t h e i r l i f e c ycle. The ph y s i c a l and b i o t i c factors involved i n the causation of t e r r i t o r i a l behaviour and the s e l e c t i o n of a t e r r i t o r y by dace were f i r s t determined through experimentation. These experiments on causation gave r i s e to hypotheses about the function of dace 3 t e r r i t o r i a l behaviour which were then tested. The r e s u l t s are r e l a t e d to work on other species and the s o c i a l organization of dace as an adaptive and adaptable system i s discussed. 4 MATERIALS AND METHODS A. Study Area The South Alouette River originates at the west end of Alouette Lake and flows westward, to P i t t River, a t r i b u t a r y of the Fraser. Figure 1 shows stations along the portion of the r i v e r from which f i s h were c o l l e c t e d . Upper reaches of the r i v e r are characterized by large pools and stretches strewn with large boulders. Lower sections l i e i n meadow land where the bottom i s composed of f i n e gravel, sand, and mud. Only the high gradient middle portion, characterized by a seri e s of fa s t flowing rapids areas with rock and gravel bottom, supports a sizeable population of longnose dace, Rhinichthys cataractae d u l c i s . Highest mean monthly flows i n the Alouette generally occur i n January and the lowest discharges i n J u l y and August. Detailed informa-t i o n on patterns of zonation of f i s h e s , temperature, discharge patterns, and gradient c h a r a c t e r i s t i c s of the South Alouette River have been pub-l i s h e d by Withler (1966), Hartman (1968), and Hartman and G i l l (1968). B. Holding Conditions C o l l e c t i o n s were made with a two-man seine (3 meshes/cm). Dace were taken from r i f f l e s by kic k i n g the substrate thus d r i v i n g f i s h e s i n t o a seine held downstream. Dace c o l l e c t e d were returned to the laboratory where they were held i n 20, 40, and 60 1 glass aquaria, large p l a s t i c containers (40 X 59 X 49 cm), and 400 1 wood and glass aquaria. A l l holding tanks contained one or more 19 cm long a i r - d i f f u s o r stone. These were located on the bottom at one end to produce a c i r c u l a r current 5 6. on a v e r t i c a l plane as described by Gee and Bartnik (1969) . The bottom of each tank was covered with a number of rocks (5-15 cm diameter). Dace were held under temperature and photoperiod regimes comparable to those i n nature. Laboratory temperature, c o n t r o l l e d by a thermostat, had a lower l i m i t of 14C. A d d i t i o n a l cooling f or holding tanks was provided by cooling c o i l s or baths. Photoperiod was regulated by a time clock connected to s i x ceiling-mounted fluorescent l i g h t f i x t u r e s each holding two 1.22 m tubes. Dace were fed Tetramin fl a k e s , frozen brine shrimp, and tubifex worms. C. Description of Stream Tank Since shallow turbulent waters make f i e l d observations nearly impossible, a flow tank was used for observation and experimentation. The stream tank was a closed system with c i r c u l a t i o n on a h o r i z o n t a l plane. The f i b e r g l a s tank consisted of two s t r a i g h t troughs (1.83 X .46 X .38 m) joined together by semi-circular elbows (0.91 m radius) at either end ( F i g . 2A) . The s t r a i g h t troughs contained glass windows (25 X 168 cm) on both sides through which observations could be made. Flow was created by four submersible water pumps each with a capacity of 2536 1 per hr. Water temperature was c o n t r o l l e d by a 7.3 m length of 2 cm diameter glass cooling c o i l located on the f l o o r of the tank. D. Experimental Channels Most experiments were c a r r i e d out i n the s t r a i g h t glass walled sections of the stream tank (Fig. 2B). With the aid of s t a i n l e s s s t e e l 7. Fi g . 2A. Top view of the stream tank showing symmetrical channels where experiments were conducted. 1 = submersible pumps; 2 = screen d i v i d e r s . F i g . 2B. Simple l i n e drawing of an observational channel of the stream tank.. 1 = screens; 2 = f a l s e bottom; 3 = concrete blocks; 4 = glass windows. Submersible pumps and cooling c o i l have been omitted. 7a. 8 screens (1.6 meshes/cm, 0.12 cm diam) and a p l e x i g l a s ' f a l s e bottom' (1.27 X 0.45 m) supported on concrete blocks, dace were confined to an 2 area of approximately 0.6 m . Water depth was adjusted to 15.2 cm and submersible pumps created an average channel v e l o c i t y of 0.6 m/sec. Except where otherwise stated, food was always a v a i l a b l e throughout the experimental channels. E. Experimental Conditions A l l breeding phase experiments were conducted under a 16 hr l i g h t -8 hr dark photoperiod and with 14-19C temperatures. A 12 hr l i g h t - 12 hr dark photoperiod and 13-15C temperatures were maintained f o r l a t e summer experiments. During winter r e p l i c a t e s , an 8 hr l i g h t - 16 hr dark photo-period and 6-10C temperatures pre v a i l e d . Densities of dace encountered i n nature were used as guidelines f o r experimental d e n s i t i e s . D a i l y photoperiod was regulated by a time clock. Daylight i l l u m i n a -t i o n was provided by s i x ceiling-mounted fluorescent l i g h t f i x t u r e s each holding two 1.22 m tubes. During the night period, the tank was i l l u m i n a -ted by four 60-w red incandescent l i g h t bulbs held i n gooseneck lamps. Observations were e i t h e r immediately written down or tape recorded and l a t e r transferred to data sheets. Day observations always were made both before and a f t e r night periods. Special experimental environments and methods w i l l be described i n s p e c i f i c experimental sections. 9 F. Experimental Fi s h Dace i n breeding condition were c o l l e c t e d from the Alouette River during the spring breeding period. This seasonal constraint along with • the r e l a t i v e l y long periods of time required for experiment r e p l i c a t i o n did not allow observation of large numbers of s i m i l a r animals. Instead, r e l a t i v e l y few i n d i v i d u a l s were studied i n t e n s i v e l y . Non-breeding dace for l a t e summer experiments were c o l l e c t e d during the f i r s t week of September, while dace f o r winter r e p l i c a t e s were taken i n January. Adult longnose dace from the Mink River, Manitoba, were c o l l e c t e d by Dr. J.H. Gee, Un i v e r s i t y of Manitoba, and flown to the Un i v e r s i t y of B r i t i s h Columbia, Vancouver. Dace usually were discarded at the completion of each experiment. However, a f t e r some experiments ( i . e . , breeding phase experiments) dace were returned to holding tanks and used i n l a t e r experiments i n which the experimental h i s t o r y of the te s t f i s h was i r r e l e v a n t . Both f i e l d and laboratory f i s h retained for stomach analyses were k i l l e d immediately by immersion i n 10% formalin. They were dissected l a t e r . Small f i n c l i p s on d i f f e r e n t regions of the caudal f i n were used so that i n d i v i d u a l s could be i d e n t i f i e d during observations?;. Dace marked by t h i s method showed no observable differences i n behaviour from non-clipped f i s h . A l l f i s h used f or breeding experiments were tested for ripeness to ensure that the condition of f i s h was r e l a t i v e l y constant between r e p l i c a t e s . Female dace used for spawning experiments were checked f o r ovulation by stroking the abdomen backwards. Breeding dace were sexed by abdomen s i z e , presence of tuberculation, and e a s i l y extruded 10 sex products. Sexing of non-breeding dace, however, r e l i e d on the sexually dimorphic t r a i t of pectoral f i n length. Male p e c t o r a l length i s greater than that of females, but occasional exceptions e x i s t . For th i s reason, dissections for sex v e r i f i c a t i o n were performed once ex-periments were terminated. However, a l l such autopsies confirmed external sexing. 11 SECTION 1 THE SOCIAL BEHAVIOUR OF LONGNOSE DACE A. Desc r i p t i o n of the Behaviour of R. c_. d u l c i s a. Reproductive Behaviour Breeding longnose males defend t e r r i t o r i e s (approx. 10 cm i n diameter) into which they entice females to spawn. Spawning occurs i n r i f f l e s where the substrate i s coarse and provides natural depressions between pieces of substrate f o r egg deposition. Under simulated r i f f l e conditions, R. c_. d u l c i s males and females display most ag o n i s t i c and courtship a c t i v i t y during the night (Fig. 3), and spawning occurs under darkness ( F i g . 4). I n i t i a l l y , males court females entering t h e i r t e r r i t o r i e s , by 'nudging' and 'following'. T e r r i t o r i a l males also frequently perform 'substrate probing' during which the snout i s pushed between pieces of substrate. Although males perform substrate probing i n the absence of females, i t i s i n t e n s i f i e d once a female enters the t e r r i t o r y . Because male dace perform t h i s behaviour pattern i n the absence of females, i t has been suggested that i n addition to being a v i t a l l i n k i n the courtship sequence ( i . e . , nest s i t e demonstration) this behaviour pattern also may be important i n nest s i t e preparation (Bartnik, MS, 1970). Once the female has entered the t e r r i t o r y , males further court females by 'trembling' during which males v i b r a t e t h e i r bodies at high frequencies. Males, however, may react aggressively to females which f a i l to respond to courtship within the t e r r i t o r y . Ripe male dace without t e r r i t o r i e s dqy wwawwrnnishi JMHIWM dav v.tbimM n.aht-mmmm 357 Fig. 3. D i e l p e r i o d i c i t y of agonistic and courtship a c t i v i t y of R. c_. d u l c i s as recorded i n the stream tank. A t o t a l of 8 dace (4 female, 4 male) were tested. (LD 16:8) 13 F i g . 4. Time i n t e r v a l s over which dace spawnings were observed ( s o l i d l i n e s ) or occurred but were not observed (broken l i n e s ) . The f i n d i n g of eggs i n the tank v e r i f i e d un-observed spawnings. Numbers i n parentheses i n d i c a t e the number of spawnings performed over that i n t e r v a l of time. (LD 16:8). Data from Bartnik (1970, MS, 1972). A t o t a l of ten R. c_. cataractae ( f i v e female, f i v e male) and 36 R. c. dul cxs (18 female, 18 male) were tested. 14 p e r s i s t e n t l y follow, nudge, and 'quiver' against females i n an attempt to spawn with them. Females, however, never respond to such courtship from n o n - t e r r i t o r i a l males. Once females are receptive, they enter the male's t e r r i t o r y and probe the substrate i n the same manner as males. The male often quivers p a r a l l e l to the female as she does so. Both f i s h then assume a p o s i t i o n on the bottom over the depression they have probed and go through a spawn-ing act i n which both quiver f or 1-2 sec. Eggs and m i l t are released and the f e r t i l i z e d eggs f a l l between rocks, adhering to the surface of under-l y i n g stones. This concludes the spawning sequence and the female leaves the nest s i t e . Once spawning i s complete, the parental male displays a high degree of s i t e attachment. He remains d i r e c t l y over the nest s i t e , probes the substrate frequently, and defends the area against a l l f i s h with the ex-ception of receptive females with which he may spawn. The term "parental" males used herein re f e r s to t e r r i t o r i a l males with eggs. Males spawn with several females or the same female a number of times. b. Agonistic Behaviour Patterns Recorded During Observations Butt: The f i s h thrusts with the snout or side of the head. Observed i n both sexes. B i t e : One f i s h quickly closes i t s mouth over some part of the body of a second f i s h . Observed i n both sexes. Dart: One f i s h swims r a p i d l y toward a second f i s h . Observed i n both sexes but performed more frequently by males. 15 Chase: One f i s h follows a second f i s h c l o s e l y , b i t i n g whenever the f l e e i n g f i s h slows or stops. Ob-served i n both sexes. Fight: Two f i s h p a r a l l e l to one another, with t h e i r heads together, go through a serie s of quick, sweeping, l a t e r a l head butts, the majority of which are glancing blows causing the f i s h to sli d e , over or under the. opponent's head. When thi s crossing over takes place combatants have merely reversed posi t i o n s and they immediately butt each other again. Observed i n both sexes. c. Courtship Behaviour Patterns Recorded During Observations Follow: A male swims behind a female without making contact. Nudge: In l a t e r a l nudging, a male while p a r a l l e l to a female, pushes h i s side against that of the female i n a swaying motion. In nose-nudging, a male pushes h i s snout against the abdomen of a female. Nibble: A male gently opens and closes i t s mouth on the dorsal f i n , caudal f i n , or dorsal surface of the head of a female. 16 Substrate Probing: The f i s h pushes i t s snout, i n a quivering motion, between pieces of substrate ; the long axis of the body making an angle of about 45-90° with the bottom. Although the or i e n t a t i o n of the body i s often s i m i l a r during feeding acts, the d i s t i n c t i v e quivering motion i s lacking. Observed i n both sexes but per-formed more frequently by males. Quiver: While p a r a l l e l to a female, the male goes through a number of f o r c e f u l muscular contractions which cause l a t e r a l undulations to pass down the length of the body. Females perform quivering only during actual spawning. Tremble: The tremble i s d i f f e r e n t from the quiver and . i s performed only by males. The male goes through a serie s of high frequency v i b r a t i o n s i n which the ent i r e body trembles. These trembles l a s t 1/2-2 sec and are repeated every 1/2-1 sec i n the presence of a female. d. Egg Eating I n h i b i t i o n i . Outline of Experiment The term "egg eating i n h i b i t i o n " has been used i n the behavioural literafeuree with respect to a parental state during which f i s h e s are i n h i b i t e d from eating eggs. Since parental longnose males have never 17 been observed to eat the eggs they guarded e i t h e r i n the laboratory or f i e l d , i t seems l i k e l y that males are i n h i b i t e d from eating t h e i r own eggs. Induction of a state of egg eating i n h i b i t i o n by the presence of eggs and recent spawning experience has been observed i n the stickleback (Van I e r s e l , 1953) and the blue gourami (Kramer and L i l e y , 1971). To determine i f such a phenomenon e x i s t s i n dace, t e r r i t o r i a l males with both eggs and recent spawning experience and t e r r i t o r i a l and n o n - t e r r i -t o r i a l males without e i t h e r eggs or recent spawning experience, were exposed to recently f e r t i l i z e d eggs. Numbers of eggs eaten by experimental dace were determined by d i r e c t observation and stomach analyses. i i . Procedure Tests were c a r r i e d out i n pa i r s w i t h i n two p a r a l l e l channels (60 X 23 cm) separated by a black p l e x i g l a s d i v i d e r . The bottom of each channel 2 was covered with f i n e gravel (0.5-1.0 cm diam) except for a 10 cm patch of coarse gravel (3-5 cm diam) s u i t a b l e f o r spawning. A small enclosure 3 (approx. 1000 cm ) made from f l a t rocks and consisting of three v e r t i c a l walls and a h o r i z o n t a l roof was positioned over t h i s coarse gravel. Dace were attracted to such enclosures and took up residence i n them. One channel held a parental male with e i t h e r 3,12, 24, or 72 hr having elapsed since he spawned. The other channel held a non-parental male. Parental males were provided by allowing males to spawn with a female i n the coarse gravel under the enclosure. Females were removed immediately a f t e r spawning. The eggs spawned by the female were l e f t i n the gravel where they were guarded by the parental male. T e r r i t o r i a l and 18 n o n - t e r r i t o r i a l types of males were distinguished by the b r i e f i n t r o d u c t i o n of a second (intruder) male. T e r r i t o r i a l males defended the enclosure while n o n - t e r r i t o r i a l ones allowed the intruder to enter i t and share i t . Each male was presented with 100 eggs from a batch produced through a r t i f i -c i a l f e r t i l i z a t i o n . Presented eggs were a l l v i a b l e and batches varied i n age from 12 to 24 hr. Eggs from the same batch were used for each pair of test dace. Eggs were presented by means of a large-bore eyedropper, a f t e r water v e l o c i t y was reduced to prevent d r i f t . Eggs were dropped d i r e c t l y i n t o the enclosures. Although some eggs f e l l between crevices i n the sub-s t r a t e and became i n a c e s s i b l e , a large number remained a v a i l a b l e to the f i s h i n each t e s t . A f t e r a 3 hr period of i r r e g u l a r l y spaced observations, dace were removed and k i l l e d . Their stomachs were examined l a t e r . i i i . Experimental F i s h Eight males, dace measuring 87 to 101 mm i n fork length were tested for egg eating i n h i b i t i o n . i v . Results and Discussion Some eggs were eaten by dace i n each of the four t e s t s (Table I ) . Parental males tested 3, 12, and 24 hr a f t e r spawning did not eat any eggs although each was observed to probe the substrate where eggs were present. Only the parental male with spawning experience 72 hr before egg presenta-t i o n , ate eggs. This parental male no longer defended h i s nest s i t e and was c l a s s i f i e d as a n o n - t e r r i t o r i a l parental male. A l l other t e r r i t o r i a l and n o n - t e r r i t o r i a l dace ate some of the eggs presented (Table I ) . These r e s u l t s agree with l a t e r experiments (Tables XII, XIII and XIV)in which 19 Table I. Number of eggs eaten by d i f f e r e n t types of dace presented with recently f e r t i l i z e d eggs Number of Eggs Eaten Parental Male Non-Parental Male (Hours a f t e r spawning) ^ Egg Batch 3hr 12hr 24hr 72hr t e r r i t o r i a l n o n - t e r r i t o r i a l 1- 0 10 2- 0 2 3- 0 11 4- 6 7 n o n - t e r r i t o r i a l parental male with spawning experience 72 hr before egg presentation 20 parental males did not eat eggs while t e r r i t o r i a l and n o n - t e r r i t o r i a l ones did. The r e s u l t s i n d i c a t e that parental male dace display egg eating i n h i b i t i o n f or up to a day a f t e r spawning. Existence of the egg eating i n h i b i t i o n phenomenon would prevent egg predation between c l o s e l y grouped parental male dace where the spread of eggs from neighbouring nest s i t e s might overlap. In three=spined sticklebacks, egg eating i n h i b i t i o n i s not place s p e c i f i c , since nest r a i d i n g males do not eat stolen eggs but carry them back to t h e i r own nest (Wootton, 1971a). Whether egg eating i n h i b i -t i o n i n parental male longnose dace i s s i t e s p e c i f i c i s not known. However, this would appear to be somewhat i r r e l e v a n t since nest s i t e t enacity i s strongest at t h i s time. e. Comparison with R. c_. cataractae The longnose dace, Rhinichthys cataractae, forms a number of geographic races whose su b - s p e c i f i c status i s i n doubt (McPhail and Lindsey, 1970). Two such sub-species are R. cataractae cataractae (Valenciennes), which occurs east of the Continental Divide, and R. cataractae d u l c i s ( Girard), which occurs on both sides of the Continental Divide (Jordan and Evermann, 1896). For purposes of convenience, Mink River, Manitoba, and Alouette River, B r i t i s h Columbia, populations of longnose dace are r e f e r r e d to i n the present study as sub-species cataractae and d u l c i s r e s p e c t i v e l y . However, i t should be understood that when I r e f e r to behavioural differences between R. c_. cataractae and R. _c. d u l c i s , the data and conclusions apply only to my 21 population samples and are not n e c e s s a r i l y sub-species s p e c i f i c . Observa-tions of more populations of each sub-species over t h e i r geographic ranges w i l l be necessary before we can know whether r e s u l t s given here apply to d i f f e r e n t populations. The reproductive biology of the eastern sub-species, R. c_. cataractae, has been described (Bartnik, 1970). But l i t t l e was known of the reproductive habits of the western sub-species, R. c_. d u l c i s , u n t i l the present study. Under simulated r i f f l e conditions, observations of breeding R. c_. d u l c i s from Alouette River revealed that although patterns of breeding behaviour of the two sub-species are almost i d e n t i c a l , there are s t r i k i n g differences i n phenotypic appearance, d i u r n a l rhythm of breeding a c t i v i t y , and female t e r r i t o r i a l i t y . Unlike R. £. d u l c i s , R. c_. cataractae are reproductively active during daylight hours ( F i g . 4), and males display bright crimson patches on the v e n t r a l surface ( F i g . 5) which function as a v i s u a l cue for sex d i s c r i m i n a t i o n (Bartnik, MS., 1970). Breeding c o l o r a t i o n i n d u l c i s males i s f a i n t or absent ( F i g . 5), and i t seems u n l i k e l y that the difference i n c o l o r a t i o n forms the basis for sexual d i s c r i m i n a t i o n i n e i t h e r sex under night condi-tions . The difference i n time of breeding of the two sub-species (Fig. 4) continues to be puzzling. One s e l e c t i o n pressure suggested which may have resulted i n nocturnal breeding i n R. c_. d u l c i s i s that due to predation by sc u l p i n s . Cottus sp., (Bartnik, 1972). But f i e l d evidence does not support the suggestion, since of 38 s c u l p i n stomachs examined none contained 22 F i g . 5. Rhinichthys cataractae cataractae and R. c_. d u l c i s males from Mink River, Manitoba, and Alouette River, B r i t i s h Columbia, r e s p e c t i v e l y . Arranged i n order from top to bottom are l a t e r a l view of R. c_. cataractae male, v e n t r a l view of same male, l a t e r a l view of R. c_. d u l c i s male, and ve n t r a l view of same male. 23 dace. However, studies of the r o l e of predation i n the evolution of f i s h species often lack both d i r e c t observation of predation and recoveries of the evolving prey from predator stomachs (McPhail, 1969). Eight j u v e n i l e steelhead trout, Salmo g a i r d n e r i , dissected also produced no dace, although one trout stomach did contain s i x f i s h i d e n t i f i e d as salmonid f r y . Infor-mation on dace egg predation and observations on behavioural i n t e r a c t i o n s between j u v e n i l e steelhead trout, s c u l p i n s , and dace are presented i n a l a t e r section. Another difference between the two sub-species i s the distance at which males f i r s t begin to court females. During R. c_. d u l c i s courtship, the distance between male and female i s almost always les s than 5 cm.R. c_. cataractae males, however, generally begin to court females at a distance of up to 12 cm. These s l i g h t q u a l i t a t i v e differences i n breeding behaviour may be a t t r i b u t e d to the presence or absence of daylight. Agonistic be-haviour patterns performed by R. c_. cataractae and R. £. d u l c i s during defence of t e r r i t o r y are e s s e n t i a l l y i d e n t i c a l . By f a r the most i n t e r e s t i n g difference to be exposed by the comparison of Alouette and Mink r i v e r populations of longnose dace deals with female t e r r i t o r i a l i t y . During the breeding period, only male R. c_. cataractae vigorously defend t e r r i t o r i e s , while females are more r e t i r i n g (Bartnik, 1970). In sharp contrast, both male and female R_. c_. d u l c i s a c t i v e l y defend t e r r i t o r i e s before and a f t e r breeding. Agonistic behaviour patterns observed only i n males of cataractae are performed by both sexes of d u l c i s (Table I I ) . 24 Table I I . Agonistic behaviour patterns commonly performed (X) by R. c_. cataractae and R. c_. d u l c i s during the breeding phase. Behaviour Pattern R. c_. cataractae R. £. d u l c i s mallej-.;-. f emal-e fhal€jc female Butt X - X X Bi t e X X X Dart X - X X Chase X - X X Fight X X X During the breeding phase, t e r r i t o r i a l i t y p e r s i s t s even i n d u l c i s females that are shown by d i s s e c t i o n to be completely spent. Intensive t e r r i t o r i a l behaviour by male dace, however, i s r e s t r i c t e d to r i p e i n d i v i -duals. A comparison of the two populations of longnose daee described here, i n which both males and females are t e r r i t o r i a l i n one and only males i n the other, may be useful i n providing information about the causa-t i o n and possibly the function of breeding t e r r i t o r y . I t i s suggested that this difference i n female t e r r i t o r i a l i t y between the two sub-species (R. £. d u l c i s from Alouette River, B r i t i s h Columbia and R. c_. cataractae from Mink River, Manitoba) i s r e l a t e d to differences i n adult d i s t r i b u t i o n during the breeding phase. A recent study by Gibbons and Gee (1972) of seasonal d i s t r i b u t i o n and abundance changes i n R. c_. cataractae i n Mink River, Manitoba, has documented the segregation of adult 25 male and female dace into d i f f e r e n t environments during pre-breeding and breeding periods. Female dace were more abundant i n large rock r i f f l e s (substrate s i z e :>75% large rocks (>15 cm diam)) while males were predominant i n small rock r i f f l e s (substrate s i z e :>75% small rocks (5-15 cm diam)) (Fi g . 6A) . Since spawning i n the Mink River occurs p r i m a r i l y i n the small rock r i f f l e environment (Bartnik, 1970), females occupy these areas only while breeding. Outside the breeding phase, both sexes occupy the same environments. Differences i n bottom substrates of the r i f f l e environments ( i . e . , those areas where v e l o c i t y exceeds 45 cm/sec) of the Alouette and Mink r i v e r s e x i s t . Gibbons and Gee's (1972) c l a s s i f i c a t i o n scheme of the r i f f l e environments i n the Mink River i s not e n t i r e l y applicable to the Alouette River. Unlike the Mink River, rock r i f f l e areas of Alouette River are not e a s i l y d i v i s a b l e into small and large rock types. Using t h e i r c r i t e r i a , based on substrate composition, the majority of rock r i f f l e s i n Alouette River f a l l into a new category. These areas are generally heterogeneous with respect to small and large rock substrate types and homogeneous zones of e i t h e r are lacking. Such environments might be referred to as "mixed" rock r i f f l e s (substrate s i z e :<>50% large rocks (>15 cm diam) :<>50% small rocks (5-15 cm diam). A series of c o l l e c t i o n s of dace from mixed rock r i f f l e s i n Alouette River, during pre-breeding and breeding periods (March 28 - A p r i l 19, 1971), 2 revealed no s i g n i f i c a n t difference (X = 2.60, p>0.10) i n abundance of adult male and female dace (Fig. 6B). Because of the physi c a l environment, males and females occur i n close proximity. Since there i s no marked 26 1.0 0.8 0.6 <u a. ~ 0.4 o o J3 E = 0.2 A R.C.CATARACTAE 0.0 h K K a — I sma II rock riffles B R.C. DULCIS B males • females large rock riffles mixed rock riffles F i g . 6. D i s t r i b u t i o n of adult dace i n rock r i f f l e s during pre-breeding and breeding periods. (A) Densities of male, and female R. c_. cataractae i n small and large.rock r i f f l e s i n Mink River (May 23-June 15). Data from Gibb ons and Gee (1972). (B) Densities of male.and female.R. c_. d u l c i s i n mixed rock r i f f l e s i n Alouette River (March 28-April 19). 27 habitat difference between sexes during spawning, a p o t e n t i a l for frequent encounters between breeding males and unreceptive females e x i s t s . On t h i s b a s i s , the hypothesis i s proposed that the close proximity of males and females e l i c i t s t e r r i t o r i a l behaviour i n females. Experimental t e s t s of t h i s hypothesis follow i n . l a t e r sections. B. Summary and Discussion Both males and females of R . c d u l c i s occupy the "mixed" rock r i f f l e s i n Alouette River and defend t e r r i t o r i e s during the breeding phase. During the night, d u l c i s females leave t h e i r t e r r i t o r i e s to court and spawn with t e r r i t o r i a l males. A f t e r spawning, parental males ( i . e . , those with eggs) show strong nest s i t e attachment and are i n h i b i t e d from eating eggs. Males and females of R. <c. cataractae are segregated into 'small' and 'large' rock r i f f l e s i n the Mink River, and only males defend t e r r i t o r i e s . The females remain n o n - t e r r i t o r i a l and share the male-occupied small rock r i f f l e s only when taking part i n the spawning a c t i v i t i e s , which occur during the day. The d i f f e r e n t s o c i a l structures of the two populations of longnose dace described here are therefore to some extent dependent on the environ-mental conditions under which each i s found. Nowadays i t i s generally accepted that s o c i a l structure i s not a r i g i d i n s t i n c t i v e feature of species, but that i t i s quite malleable and influenced by l o c a l environmental conditions. Several studies of f i s h species (Lindroth, 1955; Kawanabe, 1957, 1958; Kalleberg, 1958; Hartman, 1965) have shown that the habit of holding t e r r i t o r i e s i n salmonids i s regu-lated and modified by the phys i c a l factors i n the stream. Recent studies of 28 i n t r a - s p e c i f i c v a r i a t i o n s i n s o c i a l organization i n ungulates (Estes, 1966) and primates (Crook, 1970) c o r r e l a t e with contrasts i n the ecology of the demes concerned. Crook (1968) has stated that e c o l o g i c a l and s o c i a l condi-tions are important i n determining whether a population does or does not e x h i b i t t e r r i t o r i a l behaviour. Gartlan and Brian (1968) found vervet monkeys to be t e r r i t o r i a l i n one habitat but to move i n groups i n another. Certain rodents may be highly t e r r i t o r i a l i n some environments, at c e r t a i n popula-t i o n concentrations, but f a i l to show such behaviour under d i f f e r e n t condi-tions (Anderson, 1961). Data on herds of g a z e l l e s , Gazella g a z e l l a , at the eastern and western ends of t h e i r range, where landscape d i f f e r s markedly, suggest how profoundly s o c i a l structure may be influenced by substrate (Klopfer, 1972). I t appears conclusive then that the p h y s i c a l and s o c i a l environment of a population may impose d i r e c t constraints on s o c i a l structure, and that behavioural c h a r a c t e r i s t i c s of species often show great phenotypic p l a s t i c i t y . Breeding t e r r i t o r i a l i t y i n both sexes ( i . e . , a heterosexual t e r r i t o r i a l system) as described here'for R. c^. d u l c i s i s a phenomenon r a r e l y described i n f i s h e s . A noteworthy exception, however, i s documentation by Morris (1958) of a heterosexual system of t e r r i t o r i e s i n the ten-spined stickleback, Pygosteus pungitius. Because the female stickleback neither nests nor per-forms parental behaviour of any kind, Morris hardly expected that she would hold a t e r r i t o r y . Yet t h i s i s what occurs; a l l the aggressive actions and postures occurring i n both sexes, i n exactly the same way. Although e n t i r e l y speculative, Morris offered a p o s s i b l e explanation f o r the existence of i a heterosexual t e r r i t o r i a l system i n Pygosteus. He suggested that since nest-29 b u i l d i n g Pygosteus males keep hidden amongst dense vegetation because of t h e i r small spines which provide an i n e f f i c i e n t anti-predator device (Hoogland e_t a l . , 1956) , the l a t e r a r r i v i n g Pygosteus females would f i n d i t d i f f i c u l t to locate these males. But, i f Pygosteus females moved into the breeding grounds with males and had no aggressive tendencies, they would be driven o f f by the males when the l a t t e r were i n t h e i r pre-sexual condition. Morris therefore concluded that the only s o l u t i o n would be f o r males and females to move into the breeding grounds together, and to both defend t e r r i t o r i e s . Although Morris did not pursue his hypothesis further, he believed that i t was quite possible that under c e r t a i n e c o l o g i c a l conditions of, say, sparse vegetation or high population density, the aggressiveness of the females of a Pygosteus population may not develop. Twelve years a f t e r Morris did h i s Pygosteus work, McKenzie and Keenleyside (1970) published a paper on a population of the North American form of Pygosteus(called Pungitius  pungitius) which provides support f o r Morris' hypothesis. Unlike the European form, the North American population . • M d nests i n r e l a t i v e l y open water away from dense plant growth. Mckenzie and Keenleyside never observed females to defend t e r r i t o r i e s i n t h i s breeding habitat. The l i k e l i -hood that female longnose dace t e r r i t o r i a l i t y serves a s i m i l a r function as that hypothesized by Morris f o r the sticklebacks ( i . e . , allows males and females to coexist within the same area) i s dealt with i n l a t e r sections. Many important ideas on the adaptiveness of s o c i a l structures have been made by induction from extensive f i e l d studies of the higher vertebrates, p a r t i c u l a r l y b i r d s and mammals. Crook's (1964, 1965), Lack's (1968) and 30 Goss-Custard's (1970) studies on avian s o c i a l systems and Crook and Gartlan's (1966) studies of primate s o c i a l systems provide examples. In these studies, possible factors suggested as s e l e c t i v e agents f o r observed s o c i a l structures include food e x p l o i t a t i o n , anti-predator defence, and the gross nature of the habitat. Some of these f a c t o r s , as they p e r t a i n to dace s o c i a l structure, are considered i n a l a t e r section of t h i s t h e s i s . 31 SECTION II FACTORS INVOLVED IN THE CAUSATION OF TERRITORIAL BEHAVIOUR AND THE SELECTION OF A TERRITORY In t h i s s ection I ask the questions: 'What causes or e l i c i t s t e r r i t o r i a l behaviour?' and 'What areas do dace sel e c t to defend?' In keeping with the e c o l o g i c a l approach of the present study, only external ( i . e . , environmental)factors are dealt with i n the i n v e s t i g a t i o n of the causation of dace t e r r i t o r i a l behaviour. Internal factors ( i . e . , hormonal) are not discussed. Presumably habitat s e l e c t i o n ( i . e . , t e r r i t o r y selection) has evolved as a means for obtaining the most favourable environment f o r s u r v i v a l and procreation. Experimental evidence e l u c i d a t i n g the relevant proximate factors involved i n habitat s e l e c t i o n by animals i s r e l a t i v e l y rare. Note-worthy exceptions are Klopfer (1963, 1965) f o r b i r d s , Wecker (1963, 1964) f o r mice, and Hughs (1966) f o r i n s e c t s . Some f i s h species have also been investigated i n t h i s regard (Kalleberg, 1958; N o r r i s , 1963; Hartman, 1965; Hagen, 1967; Sale, 1969). Both Lindroth (1955) and Hartman (1965) have stated that the behaviour of choosing and defending t e r r i t o r i e s i n f i s h appears to be a reactive type of behaviour which i s governed?: by a complex of environmental s t i m u l i . In separate experimental studies, Kalleberg (1958) and Hartman (1963) found that they could e l i c i t t e r r i t o r i a l behaviour i n young brown trout simply by presenting c e r t a i n s t i m u l i . Hartman induced trout to take up and defend p o s i t i o n s by g i v i n g them v i s u a l reference points, while Kalleberg showed that t h i s t e r r i t o r i a l behaviour was i n i t i a t e d by running water. 32 Included i n the following section are a number of experimental procedures that were designed to determine what p h y s i c a l and b i o t i c factors might be involved i n the causation of dace t e r r i t o r i a l behaviour and i n the s e l e c t i o n of t e r r i t o r i e s by male and female dace. A. Breeding Phase a. Experimental Analysis of the Factors Involved i n the Selec t i o n of T e r r i t o r i e s by Male and Female Dace The following experimentawas designed to answer several questions concerning dace t e r r i t o r i a l i t y . Breeding dace were held under test condi-tions which offered them a choice between various combinations of environ-mental features. The behavioural data gathered were thus capable of revealing the c h a r a c t e r i s t i c s of s i t e s most frequently chosen by male and female dace to e s t a b l i s h t e r r i t o r i e s . i . Outline of Experiment The experimental channel was provided with a number of structures incorporating environmental features common to rock r i f f l e s . C o l l e c t i o n s of t h i n f l a t rocks (2.5 cm high) from Alouette River, c a r e f u l l y selected f o r uniformity of s i z e , were used to make these structures. Sizes of experi-mental structures were decided upon through f i e l d observations of spaces u t i l i z e d by dace. I n d i v i d u a l structures placed i n the channel were associated with e i t h e r f i n e (0.5-1.0 cm diam) or coarse (3-5 cm diam) bottom substrate. Figure 7 shows diagramatic sketches of the d i f f e r e n t combinations of structures and bottom substrates used. Throughout the thesis each type of experimental structure i s referred to by a two l e t t e r abbreviation which 33 F i g . 7. Diagramatic sketches of s i x combinations of structures and bottom substrates placed i n the experimental channel. Accompanying abbreviations are used for reference purposes. F i r s t l e t t e r s i n abbreviations i n d i c a t e whether both overhead cover and s h e l t e r from current (B), only overhead cover (C), or only shelter from current (S) was provided. Second l e t t e r s i n d i c a t e whether the underlying substrate was coarse (C) or fi n e (F). Arrow denotes d i r e c t i o n of current. 34 indicates the features that p a r t i c u l a r structure incorporates. The f i r s t l e t t e r of each abbreviation indicates whether both overhead cover and she l t e r from current (B), only overhead cover (C), or only sh e l t e r from current (S) was provided. The second l e t t e r indicates whether the under-l y i n g substrate was coarse (C) or f i n e (F). Each experimental structure incorporated two or more environmental features. V e r t i c a l and h o r i z o n t a l rocks of BC and BF provided enclosed areas of both overhead cover and low water v e l o c i t y . Horizontal rocks of CC and CF provided overhead cover while v e r t i c a l rocks of SC and SF provided areas of low water v e l o c i t y ( i . e . , s h e l t e r from current). Table I I I l i s t s environmental features incorporated into each experimental structure. Table I I I . Environmental features incorporated into (x) experimental structures. Environmen t a l Struc ture Feature BC BF CC CF SC SF Shelter from current X X - - X X Overhead cover X X X X - -Coarse bottom substrate X - X - X -Fine bottom substrate - X - X - X Since v e r t i c a l and h o r i z o n t a l rocks were uniform i n s i z e ( F ig. 7), the area of enclosed space i n BC and BF, the area of overhead cover i n BC, BF, CC, and CF, and the depth of sheltered zone i n BC, BF, SC, and SF were 35 constant. Enclosed areas of BC and BF produced a considerable shading e f f e c t , but the amount of shading provided by the suspended overhead rocks of CC and CF was considerably l e s s . V e r t i c a l rocks of structures SC and SF produced almost no shading. In this and i n subsequent experiments the assumption was made that l e v e l s of agonistic a c t i v i t y at various areas would be i n d i c a t i v e of p r e f e r -ences f o r those areas. Preliminary tests revealed that structures providing both overhead cover and shel t e r from current (BC and BF)were occupied quickly by newly introduced dace. Thus dace crowded together i n the two enclosures provided. Under these crowded conditions, dace attempting to remove other dace from an enclosure were unable to mount a successful attack. Consequently, dace ceased to perform agonistic behaviour and the development of a competitive s i t u a t i o n was retarded. This e f f e c t was remedied by providing an addit i o n a l BF structure which reduced the crowding e f f e c t and thereby allowed i n d i v i d u a l s to e s t a b l i s h t e r r i t o r i e s more e a s i l y . Once some dace held t e r r i t o r i e s , the l e v e l of agonistic a c t i v i t y remained high and the competitive s i t u a t i o n was achieved. i i . Procedure The following procedure was repeated for each of three r e p l i c a t e s . The experimental structures with t h e i r underlying substrate types were u n i -formly spaced on the channel bottom. A l l areas between structures were covered with a layer of f i n e gravel (0.5-1.0 cm diam). In each r e p l i c a t e , s i x f i s h (three female, three male) were placed i n the channel. Following a 12 hr adjustment period, 30 min observations were made at 2 hr i n t e r v a l s over a 12-1/2 hr period each day. Four observations were made under night 36 conditions ( i . e . , red l i g h t ) and three during daylight. Night observations were more frequent since reproductive a c t i v i t y occurs at t h i s time. Number of agonistic acts won and courtship acts performed were recorded. An agonistic act won i s defined as one i n which one f i s h displays one or more agonistic behaviour patterns to a second, r e s u l t i n g i n the second f i s h or " l o s e r " leaving the area where the agonistic behaviour was performed. The "winner", however, remains at the s i t e . Although a measure of the t o t a l number of agonistic i n t e r a c t i o n s occurring at various areas would also have served to i n d i c a t e which s i t e s were most highly contested ( i . e . , preferred) by dace, the more s p e c i f i c measurement of "a g o n i s t i c acts won" was used i n t h i s and i n subsequent experiments. I t was f e l t that measuring agonistic acts won would emphasize the fact that t e r r i t o r i e s were being defended. Noble (1939) defined t e r r i t o r y as "any defended area", while Tinbergen (1957) defined t e r r i t o r i a l i t y as "a combination of i n t r a s p e c i f i c h o s t i l i t y and s i t e attachment". Therefore, dace winning agonistic bouts i n the present study were said to be t e r r i t o r i a l . Generally winners were f i s h already established at the s i t e ( i . e . , r e s i d e n t ) . Agonistic acts between unestablished dace, which often ended i n a draw with both f i s h either leaving or remaining at the s i t e , were not recorded. A courtship act re f e r s to any courtship behaviour pattern performed. Courtship a c t i v i t i e s were recorded to provide an i n d i c a t i o n of sexual motivation. Observations i n each r e p l i c a t e were terminated once the f i r s t spawning acts occurred. Only data c o l l e c t e d during the 72 hr p r i o r to spawning was used. A f t e r each r e p l i c a t e a l l gravel and rocks were removed from the channel, cleaned, and replaced. Structures were rearranged for each r e p l i c a t e . 37 i i i . Experimental F i s h Eighteen dace (nine female, nine male) measuring 86 to 101 mm i n fork length were used. A l l f i s h were held i n bare tanks for 24 hr before t e s t i n g . i v . Results and Discussion The data from a l l r e p l i c a t e s were combined and s t a t i s t i c a l analyses made with the Wilcoxon matched-pairs signed-ranks test ( S i e g e l , 1956). Since each dace was marked d i f f e r e n t l y , data f o r i n d i v i d u a l f i s h were av a i l a b l e . Therefore, i n the matched pai r s test each dace was compared against i t s e l f . The r e s u l t s show that females defended only structures which incorpor-ated both overhead cover and sheltered area components ( F i g . 8). Although such enclosures were over e i t h e r f i n e (BF) or coarse (BC) bottom substrate, one type was not defended more than (p>.05) the other ( F i g . 8). T e r r i t o r i a l dace at enclosures (BC and BF) defended the area i n s i d e the enclosure but not the area around i t . Females showed no s i g n i f i c a n t difference (p>.05) between the t o t a l number of agonistic acts won during day and night periods (Table IV). A s i g n i f i c a n t majority of these agonistic acts by females (p<.005) were against the opposite sex (Table IV). Males moved about the channel considerably more than females and thus intruded frequently on the more sedentary females who responded a g o n i s t i c a l l y . Males, however, won s i g n i f i c a n t l y more agonistic acts (p<.025) at night (Table IV). At t h i s time, three d i f f e r e n t types of structures were defended strongly by males. Each defended structure incorporated the components of overhead cover (BC and CC) and/or a sheltered area (SC). The only common component i n a l l three was coarse bottom substrate (Fig. 8). 38 F i g . 8. Mean number of a g o n i s t i c acts won per area per 30 min by breeding male and female dace during day and night. Only frequencies l a r g e r than 0.05 are shown. Replicates I-III are combined. A t o t a l of 18 dace (nine female, nine male) were tested. 39 Table IV. Mean number of agonistic acts won per 30 min by male and female dace against the same and opposite sex during day and night. Numbers i n parentheses i n d i c a t e t o t a l number of wins Replicates I-III are combined. A t o t a l of 18 dace (nine male, nine female) were tested Mean No. Agonistic Acts Won/30 min Day Night Combined by female against female 0.52 1.58 1.12 (14) (57) (71) by female against male 5.66 5.61 5.63 (153) (202) (355) by female against both female -> andemale 6.18 7.19 6.76 (167) (259) (426) by male against matbe 8.18 23.00 16.65 (221) (828) (1049) by male against female 0.92 4.13 2.76 (25) (149) (174) by male against both male and female 9.11 27.13 19.41 (246) (977) (1223) 40 A s i g n i f i c a n t l y greater number of these agonistic acts by t e r r i t o r i a l males (p=.005) were won against other males than females (Table IV). Females generally remained at BC or BF areas, while males moved about the channel competing f o r the remaining coarse substrate areas. During daylight periods, males also defended three d i f f e r e n t types of structures (BC, BF, and CC). However, at this time the only common component i n each was overhead cover (Fig. 8). Texture of bottom substrate was e i t h e r f i n e or coarse. Males r a r e l y defended BF structures at night, but defended them often during the day (Fig. 8). Although CF structures provided the overhead cover component, they were never defended ( F i g . 8). Observations showed that since t h i s s i t e was not sheltered from current, p o s i t i o n maintenance over the even surface of f i n e gravel was d i f f i c u l t . At the CC structure, however, f i s h remained stationary on the bottom with the aid of outstretched pectoral and p e l v i c f i n s placed i n crevices between pieces of coarse gravel. Dace of opposite sexes were involved i n a t o t a l of 529 agonistic acts (Table IV) of which females won 67.1% and males 32.9%. Although these r e s u l t s suggest female dominance, a close r examination of the recordings revealed that winners (either male or female) i n almost every i n t e r a c t i o n were also the resident f i s h . A resident f i s h i s defined as one which has most recently won an agonistic act at a p a r t i c u l a r s i t e and/or has spent the majority of i t s time occupying that area. The behavioural phenomena associated with p r i o r residency have been described with respect to f i s h t e r r i t o r i a l i t y by Baerends and Baerends-van Rooh (1950). That a period of p r i o r residency gives an advantage to i n d i v i d u a l f i s h i n defending t h e i r 41 t e r r i t o r y has been reported for several species of f i s h (Braddock, 1949; Chapman, 1962; Jenkins, 1969; P h i l l i p s ,1971; Myrberg, 1972). The greater win-loss r a t i o i n favour of female dace may be a t t r i b u t a b l e , therefore, to t h e i r more sedentary behaviour. The question which n a t u r a l l y follows i s whether males would display a greater preference for BC and BF areas i f they were not already occupied by t e r r i t o r i a l females. The answer i s not evident from these r e s u l t s but l a t e r experiments (see page 77 ) demonstra-ted that, although vacant enclosures were a v a i l a b l e , male dace occupied and defended open areas of coarse substrate void of any overhead structure. Enclosures (BC) were, however, usually preferred. Since dace interacted continually during each test period, win-loss records are a good i n d i c a t i o n of 1) movements of i n d i v i d u a l male and female dace, and 2) areas at which resident f i s h were most frequently challenged. A comprehensive record of agonistic a c t i v i t y of i n d i v i d u a l dace i n the ex-perimental channel i s shown i n Figure 9. The graphs i l l u s t r a t e that dace experiencing l i t t l e success i n agonistic i n t e r a c t i o n s c o n t i n u a l l y interacted with f i s h at d i f f e r e n t areas (Fig. 9). No dace remained f a i t h f u l to a par-t i c u l a r s i t e and went unchallenged. These graphs ( F i g . 9) also reveal the exchangeability of t e r r i t o r i e s . During each experiment, • dace ( p a r t i c u l a r l y males) frequently defended two or more d i f f e r e n t areas. Although the competitive nature of the test s i t u a -t i o n undoubtedly enhanced this exchangeability of t e r r i t o r i e s , occasional f i e l d observations of deserted t e r r i t o r i e s also has suggested that t h i s phenomenon e x i s t s . Symons (1971) found j u v e n i l e A t l a n t i c salmon to v o l u n t a r i l y won lost 30 -1 0 -3 0 -Ml 1 0 -< V 3 0 -c o 10 • 0) < 30 0 k_ « 10 J2 um 30 Z 10 30 10 REPLICATE J n II tn N K 4 H I of ? 1 l 6 ?2 ? 3 BC BF, BFJ CC pre 30j 10 30 10 30 101 301 10 30 10 101 BC BF, BF2 CC CF SC i>K REPLICATE [J J J L 1 289 Ql84 n n ! n • ft BC BF, :BF2 CC CF SC SF : ' tf, BF2 J Cf 30 10 301 TJ7TT 10 30 10| 301 1 1 10 30 10 10 L l vrse REPLICATE IJJ I N I U H l of o 102 o7| a D_ ?- . r CC: Cf SC 5f & CC Cf SC Si 1 "2 F i g . 9. Total number of agonistic acts won and l o s t by i n d i v i d u a l male and female dace.at d i f f e r e n t areas i n Replicates I, I I , and I I I . -p-43 s h i f t l o c a t i o n of t h e i r t e r r i t o r i e s from time to time i n an a r t i f i c i a l stream, r e s u l t i n g i n part of the population constantly being i n t r a n s i t . T e r r i t o r y locations of A s i a t i c Barbus f i s h s h i f t rather often and the same lo c a t i o n may be occupied by d i f f e r e n t males i n succession w i t h i n a few days (Kortmulder, 1972). In the f i e l d , breeding male dace have been observed at the same lo c a t i o n for up to 3 successive days. A b r i e f mark recapture experiment conducted with 25 breeding adult dace showed 15.4% of the marked f i s h to 2 be within the same m 4 days l a t e r . During the breeding phase, several areas of r i f f l e habitat i n the Alouette River were seined repeatedly u n t i l no dace were taken on f i v e successive attempts. Reseining of these same areas as l i t t l e as 4 days l a t e r often yielded d e n s i t i e s of dace equal to or exceeding those i n i t i a l l y . t a k e n . Thus movements of dace wi t h i n and probably between r i f f l e areas are common. Courtship acts were displayed s i g n i f i c a n t l y more at night by both males (p<.005) and females (p<.005; F i g . 10). Males courted females almost e x c l u s i v e l y over coarse bottom substrate. Instances of courtship by males over f i n e substrate (BF) were performed only by n o n - t e r r i t o r i a l males. Females reciprocated to courting males only at structures with coarse bottom substrate (Fig. 10). In each of the three r e p l i c a t e s , spawning occurred i n association with a d i f f e r e n t structure. The only common component i n a l l three spawning or nest s i t e s was once againscoarse bottom substrate. Unlike the de t a i l e d graphs of Figure 9, which show records of i n d i v i -dual dace, Figure 10 and the remaining graphs are presented only i n the more condensed form such as Figure 8. 44 NIGHT BF CC CF • females • males SC DAY SF F i g . 10.- Mean number of courtship acts performed per area per 30 min by male and female dace during day and night. Only frequencies l a r g e r than 0.05 are shown. R e p l i -cates I-III are combined. A t o t a l of 18 dace (nine male, nine female) were tested. 45 b. S o c i a l Factors A f f e c t i n g T e r r i t o r i a l Behaviour i n Female R. c_. d u l c i s i . Outline of Experiment Since male and female R. c_. d u l c i s occupy the same r i f f l e s during the pre-breeding and breeding periods, i t seems l i k e l y that frequent i n t e r -actions between the sexes occur. The objective of the following experiment was to determine whether such i n t e r a c t i o n between males and females acts as a causative agent for the expression of R. c_. d u l c i s female t e r r i t o r i a l i t y . Manipulations were c a r r i e d out to determine whether f i r s t the addition,and then the removal of a r i p e male brought about any s i g n i f i c a n t changes i n the l e v e l of agonistic a c t i v i t y and spacing pattern of experimental females. i i . Procedure Several adult female dace, c o l l e c t e d i n September, were held under laboratory conditions which corresponded to the changing seasons outside. Under these conditions, laboratory held females became gravid at a time corresponding to the normal f i e l d breeding phase. These females were held i n all-female groups and had had no experience of male courtship since the previous spring. Both experimental channels of the stream tank were used simultaneously. Each channel contained f i v e uniformly spaced enclosures (BC) with f i n e gravel covering the remaining bottom area. Five gravid experimental females were placed i n each of the two channels. A f t e r a 12 hr adjustment period, eight 30 min observations (four during the day and four at night) were made 46 at 2 hr i n t e r v a l s to record agonistic a c t i v i t y . In addition, the p o s i t i o n of each female was plotted at the beginning, middle, and end of each 30 min period to provide information on the spacing pattern. A f t e r the eighth observation period, a recently f i e l d - c o l l e c t e d r i p e male was added to one channel and a gravid experimental female from the lab held all-female group was added to the other channel. A further eight recordings were made. At the completion of the second day's recordings, the ripe male and gravid female added the previous day were removed from t h e i r respective channels and were placed i n the opposite channel. The addi t i o n and removal of the female provided the co n t r o l . Recordings made on the second and t h i r d days included instances of female dace abandoning enclosures a f t e r being courted by the male. The data shown i n Figure 11 were ar r i v e d at by combining the frequen-cies of occurrence of d i f f e r e n t group sizes f o r the 24 p o s i t i o n checks and then c a l c u l a t i n g the percent occurrence of each group s i z e ( i . e . % occurrence of group s i z e 1 = number of groups of s i z e 1 observed 7 t o t a l number of groups observed x 100%). When performing s t a t i s t i c a l tests on combined p o s i t i o n check data i n t h i s and i n subsequent experiments, the assumption was made that the p o s i t i o n check data were independent from one another. i i i . Experimental f i s h Twelve dace (one male, eleven female) measuring 87 to 107 mm i n fork length were used. i v . Results and Discussion The r e s u l t s indicate that the introduction of a r i p e male dace was 47 F i g . 11. Percent occurrence of d i f f e r e n t group sizes i n Channel 1 on day 1 ( f i v e female), day 2 ( f i v e female, 6ne7imale),.and day 3 ( s i x female), and i n Channel 2 on day 1 ( f i v e female), day 2 ( s i x female), and day 3 ( f i v e female, one male). F i g . 12. Total number of agonistic acts won by R. £. d u l c i s females i n Channel 1 on day 1 ( f i v e female), day 2 ( f i v e female, one male), and day 3 (s i x female), and i n Channel 2 on day 1 ( f i v e female), day 2 (six female), and day 3 ( f i v e female, one male). Numbers i n c i r c l e s indicate number of occasions female dace abandoned enclosures (BC) a f t e r being courted by the male. CHANNEL 1 CHANNEL 2 60-20-100-60-20 • 100-60-20 • 5?? 5??.lo* 6?? DAY 1 DAY 2 DAY 3 5?? 6?? 5??,lo" 2 3 3 + 1 2 Group Size 3^ c o 5 50 h 40 £ 30 c 20 o CD < 10 ..CHANNEL 1 _ 6?? ® , ° 5??.lo* /CHANNEL 2 o ' 6?? 2 DAY 48 instrumental i n bringing about a rapid change i n the spacing pattern of test females (Fig. 11). With the addition of the male to channel 1 on day 2, females were displaced from previously occupied enclosures by the courting male on 14 oecasions (Fig. 12). The previously non-aggressive females displayed agonistic behaviour patterns to other females and to the added male. As a r e s u l t of t h i s t e r r i t o r i a l behaviour, the spacing pattern of the females changed s i g n i f i c a n t l y (p<. 001) from a clumped d i s -t r i b u t i o n to a near uniform one (Fig. 11). Comparisons of spacing pat-terns are made with Contingency Chi-square tests (Si e g e l , 1956). The add i t i o n of the female to channel 2 on day 2, however, d i d not have the same e f f e c t . Although the occurrence of the larger groupings of three or more females decreased, the spacing pattern of females i n channel 2 remained clumped (p<. 001) compared with that of channel 1. Unlike the females i n channel 1, channel 2 females performed no a g o n i s t i c a c t i v i t y . However, when the male was transferred to channel 2 on day 3, the spacing pattern changed s i g n i f i c a n t l y (p<.001) as i t did when t h i s male was added to channel 1 on day 2 (Fig. 11). Channel 2 females were displaced by the courting male and the females i n turn began to defend enclosures ( F i g . 12). Other all-female groups held i n the experimental channel for up to 6 days f a i l e d to show any s i g n i f i c a n t changes i n agonistic a c t i v i t y or spacing pattern. Therefore the observed changes i n female R. c_. d u l c i s behaviour (Fig. 11 and F i g . 12) were d e f i n i t e l y r e l a t e d to the i n t e r a c t i o n with the male. When the male was removed from channel 1 and replaced with the female, the amount of agonistic a c t i v i t y ( i . e . , t e r r i t o r i a l behaviour) performed 49 decreased only s l i g h t l y (Fig. 11). However, the spacing pattern took on a more clumped form (one, two, or three f i s h per enclosure) resembling that of day 1 (p>. 05). I t i s i n t e r e s t i n g to note that i n channel 1 on day 3 the added female, which had not yet experienced cohabitation with the male, was involved i n s i x agonistic acts with other females. The f i r s t f i v e were l o s t but the s i x t h won. This observation suggests that frequent displacement by already t e r r i t o r i a l females may e l i c i t t e r r i t o r i a l behaviour i n other females. c. S o c i a l Factors A f f e c t i n g T e r r i t o r i a l Behaviour i n Female R_. c_. cataractae i . Outline of Experiment The foregoing data support the suggestion that i n t e r a c t i o n with males i s a causative agent for the expression of R. c_. d u l c i s female t e r r i t o r i a l i t y . Therefore, since R. c_. cataractae females are segregated from t h e i r males, i t seems possible that t h i s d i f f e r e n c e i n s o c i a l factors ( i . e . , no i n t e r -action with males) accounts f o r the observed diff e r e n c e i n female t e r r i t o r i -a l i t y . I f t h i s i s true, then R.' c_. cataractae females would be expected to display t e r r i t o r i a l behaviour i f they were forced to i n t e r a c t with t h e i r males. The following experiment created an a t y p i c a l grouping of breeding male and female R. c_. cataractae to determine whether the predicted response occurs. i i . Procedure Adult R. c_. cataractae were c o l l e c t e d from Mink River, Manitoba, during October and transported by a i r to the Vancouver laboratory. Dace 50 were held under three d i f f e r e n t conditions of temperature and photoperiod comparable to l a t e summer, winter, and spring (breeding) seasons (see page 8). Both sexes were kept together i n holding tanks except for the spring period when sexes were separated. Dace responded p h y s i o l o g i c a l l y to i n -creasing day length and temperatures by coming into breeding condition. Both r i p e males and gravid females (three males, three females) were placed i n an experimental channel i n which segregation of sexes was impossible. Six BC structures were uniformly spaced on the channel bottom and a l l areas between structures covered with f i n e gravel. Following a 12 hr adjustment period, 30 min observations were made at 2 hr i n t e r v a l s over a 14-1/2 hr period each day. Four observations were made under both day and night conditions. Since dace frequently moved about i n the experimental channel, i n t e r -actions between males and females were i n e v i t a b l e . For t h i s reason, experi-mental f i s h were described as being under "forced i n t e r a c t i o n " . During 3 days of such 'forced' i n t e r a c t i o n , types and frequency of a g o n i s t i c behaviour patterns performed by females were recorded. In addition, agonistic acts won by each sex and courtship a c t i v i t y performed by males which displaced females from previously occupied enclosures were scored. P o s i t i o n checks of each f i s h were made at the beginning, middle, and end of each 30 min recording period to determine occupancy rate of enclosures (BC) and "open" ( i . e . , free of structures) areas. In t h i s and i n subsequent experiments, th i s occupancy rate i s expressed as percent occurrence ( i . e . , % occurrence of dace at enclosures (BC) = number of dace occupying enclosures during the 51 day's p o s i t i o n checks -r t o t a l number of observations of dace (number:of dace x number of p o s i t i o n checks) x 100%). A f t e r the f i n a l observation on day 3, several t h i n f l a t rocks (2.5 cm high) were p i l e d i n each of two corners of the channel. These two areas provided small spaces where dace could wedge themselves and thus i s o l a t e themselves from other f i s h . Since dace could avoid i n t e r a c t i o n s with other f i s h , t h i s condition was re f e r r e d to as "not forced i n t e r a c t i o n " . Recordings were continued for t h i s fourth and f i n a l day. i i i . Experimental F i s h Six dace (three male, three female) measuring 67 to 87 mm i n fork length were used. i v . Results and Discussion Although a shortage of R. c_. cataractae females did not allow r e p l i -cation of t h i s experiment, the r e s u l t s are quite suggestive. E a r l y i n the experiment i t became obvious that a female would share an enclosure with a male, but would abandon the s i t e or t r y to defend i t should the male p e r s i s t i n courting her. The most common courtship behaviour patterns performed by males were nudging and quivering, both of which moved the female s l i g h t l y each time. During each of the f i r s t 2 days of forced i n t e r a c t i o n , female dace showed l i t t l e success i n a g o n i s t i c i n t e r a c t i o n s with males, winning only 25%. However, by the t h i r d day t h i s percentage reached 66% (Fig. 13). During the 3 days of i n t e r a c t i o n with males, females defended enclosures 2 (BC) against other females as r e a d i l y as they did against males (X = 0.28, 52 F i g . 13. Number of agonistic acts won by male and female R. £. cataractae against the same sex ( Q ) and opposite sex ( • ) during 3 days of 'forced' i n t e r a c t i o n and a fourth day not forced. Numbers i n c i r c l e s i n d i c a t e number of occasions female dace abandoned enclosures (BC) a f t e r being courted by a male. Small inset graphs i n d i c a t e percent occurrence ( E3 ) of dace at enclosures (BC), open areas (0), and i s o l a t i o n areas ( I ) . Six dace (three male, three female) were tested. 52a MALES 20 . 6 0 O 3 0 n 80 FEMALES DAY 1 BC © DAY 2 60] 30 BC 1 60 f 40 20 c o 5 < » 80 c J> 60 [ 40 201 40\ 20\ 6 0 30 1 BC ® n DAY 3 6 0 30 i BC j g . © ~ i — DAY 4 60Y 3 0 r I © 6 0 30 BC 6 0 30] BC 6 0 • 3 0 -1 BC T l O R 53 p >.50; F i g . 13). On the f i r s t day, 37.5% of a l l p o s i t i o n checks made found female dace sharing enclosures with other dace (either male or female). On days 2 and 3, however, only 4.2% of a l l such checks found th i s same s i t u a t i o n . Males occurred predominantly at enclosures (BC) which contained the appropriate spawning gravel, and agonistic i n t e r a c t i o n s between males were frequent (Fig. 13). Corresponding with the increase i n female aggressiveness on day 3 2 was a s i g n i f i c a n t increase (X =14.0, p<.001) i n the occurrence of females at enclosures compared with that of day 2 (Fig. 13). Throughout the period of forced i n t e r a c t i o n , females abandoned enclosures when c o n t i n u a l l y harassed by courting males (Fig. 13). On day 2, both displacements of females and frequency of occurrence of females at open areas peaked at 19 occasions and 76.4% res p e c t i v e l y . However, when cataractae females began to defend enclosures on day 3, the number of displacements of females by courting males decreased to 11. When defending enclosures cataractae females display agonistic behaviour patterns usually common only to cataractae males (Fig. 14). Therefore both sexes are g e n e t i c a l l y programmed f o r the f u l l complement of ago n i s t i c be-haviour patterns, but females express them only when i n t e r a c t i n g with breed-ing males. Franck (1969) suggests that behaviour patterns, instead of being g e n e t i c a l l y f u l l y reduced, may be incorporated by means of d i f f e r e n t mechanisms into a r e s e r v o i r of behaviour patterns which are l a t e n t under c e r t a i n condi-t i o n s . Present findings lend themselves w e l l to such an explanation. On day 4, the addition of i s o l a t i o n areas did l i t t l e to a l t e r the behaviour of male dace, but brought about a marked change i n female behaviour. Females almost immediately sought out small spaces i n the rock p i l e s where 54 F i g . 14. Frequency of occurrence of agonistic behaviour patterns performed .by R. c_. cataractae females during 3 days of 'forced' i n t e r a c t i o n with males. I 55 they wedged themselves. This action avoided i n t e r a c t i o n s with males and displacements of females by males were almost e n t i r e l y eliminated ( F i g . 13). Although female dace behaved d i f f e r e n t l y on days 3 and 4, a s i m i l a r reduction i n c o n f l i c t s with males was observed on both days. During day 3 of forced i n t e r a c t i o n , females were able to reduce displacement and remain r e l a t i v e l y sedentary through t e r r i t o r i a l behaviour. S i m i l a r l y , when able to s p a t i a l l y segregate themselves from males on day 4, females avoided almost a l l a g o n i s t i c a c t i v i t y and courtship harassmenfct from males. Unanswered by the two foregoing experiments on s o c i a l factors a f f e c t -ing t e r r i t o r i a l behaviour i n female dace i s the question of the genotypic or phenotypic nature of the difference between the two sub-species. Such a problem i s beyond the scope of t h i s study but the data here would suggest that females of the two populations do d i f f e r g e n e t i c a l l y . From e a r l i e r observations we know that under laboratory conditions simulating a natural r i f f l e , R. c:. d u l c i s females do not choose to i s o l a t e themselves from males, but immediately e s t a b l i s h t e r r i t o r i e s . However, the present experiment as w e l l as e a r l i e r work (Bartnik, 1970) demonstrates that R. c_. cataractae females i s o l a t e themselves from males when given the opportunity. But when unable to do so, R. c^. cataractae females w i l l e s t a b l i s h t e r r i t o r i e s . There-fore, females of the Mink River population of R. c_. cataractae exhibit con-siderable behavioural p l a s t i c i t y . The adaptation to environment i n th i s case i s phenotypic. Hopefully, further comparative studies on d i f f e r e n t populations of Rhinichthys cataractae w i l l help to support or refute the conclusions reached here. 56 d. S o c i a l Factors A f f e c t i n g T e r r i t o r i a l Behaviour i n Male R. c_. d u l c i s The r e s u l t s of the e a r l i e r experiments on factors involved i n t e r r i t o r y s e l e c t i o n ( F i g . 8) showed that during the night males defended a l l three types of structures with coarse bottom substrate. However, within i n d i v i d u a l r e p l i c a t e s only two of the three structures were defended. In each r e p l i c a t e , one structure was defended s i g n i f i c a n t l y more than the other (p<.001; Table V). Closer examination of the pattern of a c t i v i t y suggested why one p a r t i c u l a r structure was preferred over others. Figure 15 i l l u s t r a t e s f l u c t u a t i n g l e v e l s of agonistic and courtship a c t i v i t y recorded during the 72 hr before spawning at each of the preferred s t r u c -tures i n r e p l i c a t e s I, I I , and I I I . The graphs i l l u s t r a t e a strong Table V. T o t a l number of agonistic acts won by males against other males at coarse substrate structures during night Replicate Number of Agonistic Acts Won „ BC CC SC T o t a l X (Chi-square) I 21 0 231 252 p<.001 II 16 389 0 405 p<.001 II I 111 0 60 171 p<.001 66rrelaEI6n betTween l e v e l s of male and female courtship and male ag o n i s t i c a c t i v i t y . Without f a i l , increases i n courtship a c t i v i t y at a s i t e were accompanied by sharp increases i n the number of a g o n i s t i c acts won by the resident male. Replicate I (SO Replicate II (CC) *_• agonistic by d* 0- -o courtship byo^ 1 — (Courtship by°. • 119 Replicate III (BC) • J f m g M l l H Z Z Z 7 % day day • J P i g h L F i g . 15. Pattern of agonistic and courtship a c t i v i t y performed by dace during the 72 hr before spawning at preferred structures. Abbreviations i n parentheses indicate type of structure i n each r e p l i c a t e . (LD 16:8). 58 A d e t a i l e d temporal examination of a t y p i c a l sequence of agonistic and courtship a c t i v i t y at one preferred ( i . e . , highly defended) structure ( F i g . 16) i l l u s t r a t e s the r o l e of courtship by the female i n inducing the resident male to perform courtship behaviour. The subsequent increases i n agon i s t i c a c t i v i t y were due to the repeated removal of intr u d i n g males which were attracted to the s i t e . Although such a sequence of events was the general case, occasionally the female v i s i t e d a male already engaged i n defence of a s i t e . The a t t r a c t i o n of males to s i t e s where females perform courtship behaviour may a f f e c t the spacing of t e r r i t o r i a l males. This p o s s i b i l i t y i s examined i n the following experiment. e. Clustering of Male T e r r i t o r i e s Many groups of c l o s e l y spaced nests and t e r r i t o r i a l males have been located i n the Mink River (Bartnik, MS, 1970) and Alouette River (unpubl. data). S p a t i a l d i s t r i b u t i o n ( i . e . , i n ter-nest distances) of such c l u s t e r s of nests were smaller than seemed to be required by the a v a i l a b l e spawning resources. The a t t r a c t i o n of males to s i t e s where females perform court-ship behaviour, as described under the preceding heading, at f i r s t seemed to be a p l a u s i b l e explanation for the occurrence of such c l u s t e r s of t e r r i t o r i e s . However, since male dace come into breeding condition before females (Bartnik, 1970), males are usually already holding t e r r i t o r i e s when females become sexually receptive. Further experimental i n v e s t i g a t i o n of the formation and maintenance of male t e r r i t o r y c l u s t e r s follows. i . Outline of Experiment To further examine the suggestion that c l u s t e r i n g of male t e r r i t o r i e s may be a r e s u l t of female attention to p a r t i c u l a r areas, two sets of t e s t s 59 Mr-12 10h •_e Agonistic by <f o- -o Courtship by <f A-.-A Courtship by? A / K M ' y \ / K \ / \ v \ J I I L I i I I I I L I I I I I I I I I I I L K) 15 T I M E (minutes) 20 F i g . 16. Typ i c a l sequence of agonistic and courtship a c t i v i t y performed at a preferred structure. 60 were made. F i r s t l y , male dace were introduced into a pre-courtship t e s t s i t u a t i o n ( i . e . , without females) to determine the d i s t r i b u t i o n of t e r r i -t o r i e s . In a second experiment, t e r r i t o r i a l males were purposely spaced widely apart, and then a sexually responsive female was introduced to determine what influence she would have on the t e r r i t o r y spacing pattern. i i . Procedure In the f i r s t experiment, eight structures were placed on the channel bottom such as to create two zones. Four BC structures were uniformly spaced on the bottom of both upstream and downstream ends of the channel (Fig. 17A). Minimum distances between structures i n the experimental channel were comparable with distances between t e r r i t o r i a l males and nests located i n the f i e l d . A l l bottom areas between structures were covered with fine gravel. In each of three r e p l i c a t e s , four males were placed i n the channel 12 hr before the commencement of a night period. During each of the two following night periods, four equally spaced 30 min observations recorded l o c a t i o n and frequency of agonistic acts won. A f t e r the f i n a l night observation, the p o s i t i o n of each t e r r i t o r i a l male was noted. In the second experiment, the same arrangement of structures was used ( F i g . 17A), but a d i v i d i n g screen was added to separate upstream and downstream areas. In each of three r e p l i c a t e s , two males were placed i n both upstream and downstream portions of the channel. Casual observations were made over two successive night periods to v e r i f y the development of t e r r i t o r i a l behaviour and s i t e attachment. At the end of the second night, positions of t e r r i t o r i a l males were noted. Immediately before the 61 F i g . 17A. Arrangement of structures i n channel (top view). Numbers r e f e r to BC structures. Arrow denotes d i r e c t i o n of current. F i g . 17B. Pattern of a g o n i s t i c a c t i v i t y performed by t e r r i t o r i a l males during eight successive night observation periods. Four males were tested i n each r e p l i c a t e . A LTJ LH LH 0 m 0 Replicate 1 Replicate II Replicate III --i , l l 1 • • • . l l 1 I i 1 l • • • . i 1 . 1 1 | 1 . . 1 1 1 i l l 1 - • n 1 . 1 • i t . 1 . I 1 1 1 1 1 • 1 • • . • 1 2 3 4 5 6 7 8 1 2 3 4 STRUCTURES 5 6 7 8 1 2 3 4 5 6 7 8 62 commencement of the t h i r d night period, the screen d i v i d e r was removed and a recently ovulated female was added. Ovulated females almost always spawned within several hours a f t e r introduction. Observations were con-tinued for the t h i r d night. P o s i t i o n s of each male were again noted a f t e r the f i n a l observation was made. i i i . Experimental F i s h Twelve male dace, measuring 89 to 102 mm i n fork length, were used i n the f i r s t experiment. The twelve male dace used i n the second experiment measured 84 to 97 mm i n fork length. i v . Results and Discussion In the f i r s t experiment, the males held i n the experimental channel ( F i g . 17A) over two successive nights displayed an unexpected c l u s t e r i n g of t e r r i t o r i e s . Actual mean distance between t e r r i t o r i e s i n two r e p l i c a t e s was equivalent to the minimum mean distance attainable. For the t h i r d r eplicate, i t was considerably l e s s than the maximum distance (Table VI). Although female courtship or attention, thought to be instrumental i n the a t t r a c t i o n of males to l o c a l i z e d areas, had been eliminated, the c l u s t e r i n g e f f e c t s t i l l occurred. The importance of male i n t e r a c t i o n i n the formation of t e r r i t o r y c l u s t e r s became most evident during the f i r s t experiment. Formation of c l u s t e r s consisted of a trespassing or i n t r u d i n g male i n t e r a c t i n g with a newly established t e r r i t o r i a l male. The aggressive behaviour of the t e r r i t o r i a l male not only r e p e l l e d the trespassing male from the t e r r i t o r y , but also e l i c i t e d t e r r i t o r i a l behaviour i n the trespassing f i s h . Consequently, the trespassing male began to defend a nearby s i t e against other males as 63 Table VI. Actual mean distance between four t e r r i t o r i a l male dace as compared with maximum and minimum mean distances attainable. Distances are measured from centers of each BC structure. Replicate Mean Distance (cm) Actual Maximum Minimum I 23 107.3 23 II 23 107.3 23 III 70.5 107.3 23 Table VII. Actual mean distance between four t e r r i t o r i a l male dace as compared with maximum and minimum mean distances ' attainable before and a f t e r screen d i v i d e r was removed and a female was introduced WITH SCREEN (NO FEMALE) NO SCREEN (WITH FEMALE) Mean Distance (cm) Mean Distance (cm) Replicate Actual Maximum Minimum Actual Maximum Minimum I 95.5 107.3 80.6 95.5 107.3 23 II 100.6 107.3 80.6 100.6 107.3 23 III 87.3 107.3 80.6 94.0 10.7.3 23 64 w e l l as the I n i t i a l t e r r i t o r i a l male. Two males engaged i n agonistic a c t i v i t y attracted other males which also interacted with them. Therefore, i t appeared that the e f f e c t of this behaviour was not to increase the d i s -tance between males, but to a t t r a c t other males td the v i c i n i t y of t e r r i -t o r i a l males. During the f i r s t f i v e observations, t e r r i t o r i a l males of r e p l i c a t e s I, I I , and III won an average of 10.2 agonistic acts each (Fig. 17B). However, this l e v e l dropped to 5.6 agonistic acts won per t e r r i t o r i a l male for the l a s t three observations. The o v e r a l l amount of agonistic a c t i v i t y d i d not change dramatically from the f i r s t to the second night, but t h i s a c t i v i t y was d i s t r i b u t e d between more t e r r i t o r i a l males during the second night ( F i g . 17B). The observed reduction i n f i g h t i n g by i n d i v i d u a l males was r e l a t e d to the r e s t r i c t i o n of male a c t i v i t y to the t e r r i t o r y . A f t e r a c q u i s i t i o n of a t e r r i t o r y , dace began to confine t h e i r movements to a l i m i t e d area and antagonism between neighbours was reduced. Males of one r e p l i c a t e , l e f t i n the channel a t h i r d night, continued to i n t e r a c t at t h i s lower l e v e l . With the addition of the screen p a r t i t i o n i n the second experiment, a more widely spaced d i s t r i b u t i o n of male t e r r i t o r i e s was created than that which occurred i n the unscreened channel (compare actual mean distances i n Table VI and Table VII). A f t e r removal of the screen and addition of an ovulated female, the d i s t r i b u t i o n of dace remained e s s e n t i a l l y the same (Table VII). Only i n r e p l i c a t e I I I did a s h i f t occur. I t involved one male and amounted to an increase i n the actual mean distance to 94 cm from 87.3 cm. 65 In each r e p l i c a t e , the ovulated female swam throughout the channel encountering a l l males before r e s t r i c t i n g the majority of her courtship a c t i v i t y to one p a r t i c u l a r male. A f t e r each v i s u a l and/or p h y s i c a l con-tact made with the female, t e r r i t o r i a l males immediately commenced sub-st r a t e probing i n t h e i r t e r r i t o r i e s . A male that had strayed outside i t s t e r r i t o r y quickly returned to i t when a female entered the v i c i n i t y . Unexpectedly, other t e r r i t o r i a l males were not attracted to the s i t e where the female performed courtship behaviour. Instead, t e r r i t o r i a l males re-mained f a i t h f u l to t h e i r t e r r i t o r i e s and almost no rearrangement of t e r r i -tory spacing occurred. As a r e s u l t , a courting male received l i t t l e i n -terference from neighbouring t e r r i t o r i a l males. In e a r l i e r experiments (see page 58 ), which suggested female attention to p a r t i c u l a r s i t e s a t t r a c t e d other males, both sexes were always placed into the tank simul-taneously. Unlike i n the present experiment, male t e r r i t o r i e s were not yet established and/or stable i n those e a r l i e r experiments. Under those un-stable conditions, males followed females and aggregated around s i t e s at which females performed courtship behaviour. These males often occupied and defended s u i t a b l e areas near by. Since males generally precede females i n breeding readiness, the s i t u a t i o n described i n the present experiment would appear to be more comparable with f i e l d conditions. The present findings show that r i p e male dace are attracted to and i n t e r a c t with other males r e s u l t i n g i n the formation of t e r r i t o r y c l u s t e r s . As each i n t e r a c t i n g male claims a t e r r i t o r y , the agonistic i n t e r a c t i o n s become l i m i t e d to the defence of the t e r r i t o r y and l e v e l s of a g o n i s t i c a c t i v i t y shown by i n d i v i d u a l males decrease. In the presence of a receptive 66 female, t e r r i t o r i a l males of such c l u s t e r s remain f a i t h f u l to t h e i r chosen t e r r i t o r i e s and intrude l i t t l e upon neighbouring f i s h involved i n court-ship and spawning. Once these males spawn they show even stronger nest s i t e t e nacity, thus enhancing the s t a b i l i t y of the assemblage. Many b i r d species gather on arenas or leks where males compete for females, e.g., the r u f f , grouse, and blackcock. Male longear sunfish con-gregate along r i v e r edges and nests are grouped c l o s e l y together i n colonies (Keenleyside, 1970). Each nest i s guarded by a strongly t e r r i t o r i a l male. On the other hand, van den Assem (1967) and Jenni (1972) report that ex-perimental p a i r s of male sticklebacks space themselves out widely i n large tanks (100 x 600 cm) by s e l e c t i n g nest s i t e s at an average distance of 85% and 83 to 96% r e s p e c t i v e l y oftthe maximum possible distance from r i v a l nests. f. Behaviour of Clustered T e r r i t o r i a l Male Dace i . Outline of Experiment It seems reasonable to i n f e r from the foregoing data that the beha-viour shown by t e r r i t o r i a l male dace assembled close together may have s e l e c t i v e value. The following tests were conducted to determine whether any behavioural differences between clustered males and more i s o l a t e d ones e x i s t . An obvious difference between the two types i s i n the amount of i n t e r a c t i o n each might experience with other males. The foregoing experi-ment suggests that clustered t e r r i t o r i a l males which i n t e r a c t frequently with other dace might remain f a i t h f u l to t h e i r chosen s i t e s longer ( i . e . , be l e s s l i k e l y to abandon a t e r r i t o r y or nest s i t e ) than would i s o l a t e d males. 67 In the following experiment the behaviour of 1) parental males experiencing frequent in t e r a c t i o n s with other males, and 2) parental males which do not experience such i n t e r a c t i o n i s compared. i i . Procedure T e r r i t o r i a l males with eggs were provided by introducing a s i n g l e male i n t o a small sectioned off area of the experimental channel (60 x 23 cm) from which no other f i s h were v i s i b l e . A BC structure was located at one end and a BF structure at the opposite end. The remaining bottom area was covered with f i n e gravel. The BC structure provided the necessary spawning substrate while the BF structure acted as an alternate s i t e , providing the same environmental features except for coarse gravel. At the commencement of the night period, 12 hr a f t e r the male's introduction, an ovulated female was added to the channel. Spawning i n v a r i a b l y took place at the BC area with several spawning acts occurring. Once the female's abdomen was noticeably reduced i n s i z e , she was removed without dis t u r b i n g the male. Two treatments were used for such males with eggs. One set of three r e p l i c a t e s l e f t the male unmolested for 83 to 90 hr, while making 12 to 14 i r r e g u l a r l y spaced 15 min recordings of substrate probing frequency and time spent on the nest. Although a consistent spacing of recordings between r e p l i c a t e s i s preferable for comparative purposes, the fact that spawnings occurred at d i f f e r e n t times of the night made t h i s d i f f i c u l t to achieve. In the second set of r e p l i c a t e s , the t e r r i t o r i a l male was allowed to i n t e r a c t with intruding males at i r r e g u l a r l y spaced i n t e r v a l s for three or 68 more days. Immediately a f t e r a 15 min recording period, the top (horizon-t a l rock) of the BF structure was removed. This was done so that when a r i p e male (intruder) was added to the channel i t would seek out the only available cover ( i . e . , the BC structure-nest s i t e ) , attempt to enter that enclosure, and encounter the parental male. During any one i n t e r a c t i o n period, a t o t a l of f i v e i n t e r a c t i o n s were allowed between the parental male and the intruder male. For each i n t e r a c t i o n , the time between i n t r u s i o n and a g o n i s t i c response of the parental male ( i . e . , latency to attack) was measured. Since male intruders sometimes became reluctant to enter the nest s i t e a f t e r being attacked by the defending parental male, i t was necessary to replace such intruders with another male i n order to accumulate f i v e i n t e r a c t i o n s . Therefore, during each r e p l i c a t e , several d i f f e r e n t in t r u d e r r males were used. A f t e r the f i f t h i n t e r a c t i o n , the intruder male was removed and the.top of the BF structure was replaced. Although a t o t a l of 27 i n t e r a c t i o n bouts were staged, only i n 24 bouts did f i v e successive i n t e r a c t i o n s take place between the t e r r i t o r i a l male and the intruder; In the other three bouts, which a l l took place l a t e i n the t e s t period, the f i v e i n t e r a c t i o n s did not occur e i t h e r because the t e r r i t o r i a l male f a i l e d to respond aggressively to the intruder or because he merely abandoned the enclosure a f t e r the intruder entered i t . Immediately a f t e r each i n t e r a c t i o n period, a f i n a l 15 min observation was made to once again record substrate probes and time spent on the nest by the parental male. I t was f e l t that the above described procedure would best mimic the s i t u a t i o n i n which a t e r r i t o r i a l resident would i n t e r a c t with adjacently 69 t e r r i t o r i a l males or intruders over intermittent periods. Unlike mirror images, confined l i v e c o n s p e c i f i c s , or models which can neither i n t e r a c t with nor f l e e from test f i s h , the present test conditions provided a r e a l i s t i c analog of a t e r r i t o r i a l i n t r u s i o n . i i i . Experimental Fish A t o t a l of 16 males were used. Parental males measured 90 to 101 mm i n fork length while intruder males measured 87 to 99 mm i n fork length. i v . Results and Discussion Although a c e r t a i n amount of v a r i a b i l i t y occurred between r e p l i c a t e s within each treatment, d i f f e r e n t trends were s t i l l evident i n each of the two treatments. Parental males i n t e r a c t i n g with conspecifics displayed a high degree of s i t e attachment as indicated by the prolonged period of s i t e f a i t h f u l n e s s ( i . e . , time spent on nest) (Fig. 18). Twenty-four hr a f t e r spawning a l l i n t e r a c t i n g parental males s t i l l spent 100% or nearly 100% of t h e i r time over the nest s i t e , but males with no i n t e r a c t i o n were already absent from t h e i r nest s i t e s for short i n t e r v a l s . These l a t t e r males were usually located at the alternate enclosure (BE). Frequency of substrate probing i n both groups was at a peak immediately following spawning, but declined within several hours (Fig. 18). Interacting parental males continued to probe the nest substrate throughout the test period. Substrate probing a c t i v i t y i n parental males without i n t e r a c t i o n , however, usually became n i l within 24 hr of spawning (Fig. 18). Parental males always performed more substrate probing during the period following an i n t e r a c t i o n compared to the period preceding i t . A f t e r 70 F i g . 18. Frequency.of substrate probing ( s o l i d l i n e s ) and time spent on nest, (broken l i n e s ) by parental males with and without i n t e r a c t i o n with con s p e c i f i c males. Points " connected by dotted l i n e s are the 15 min periods before and a f t e r i n t e r a c t i o n with intruding males. 20 0 20 0 40 20 0 20 0 40 20 0 40 20 0 70a I N T E R A C T I O N J 1 1 1 I I I I I L J I I I I I I I I L_ 10 20 30 40 50 60 70 80 90 HOURS AFTER SPAWNING 71 i n t e r a c t i n g with intruder males, parental males also spent more time over the nest ( F i g . 18). For the f i r s t i n t e r a c t i o n i n each s e r i e s , parental males were slow i n responding aggressively to intruders. How-ever, the latency period become progressively shorter a f t e r the f i r s t i n t e r a c t i o n ( F ig. 19). In both i n t e r a c t i o n and no i n t e r a c t i o n groups, parental male dace oc c a s i o n a l l y l e f t the nest s i t e f o r short periods of 1-3 sec shor t l y a f t e r spawning. During these movements the male moved approximately 5-10 cm from the t e r r i t o r y , only to quickly return to the nest s i t e again. Such b r i e f movements i n nature may expose males to nearby females and/or bring the parental male into v i s u a l contact . with.other t e r r i t o r i a l males. Possi b l e e f f e c t s of such movements, are increased sexual and aggressive arousal of both the parental male and his neighbours. Although the sample s i z e i s very small, the r e s u l t s i n d i c a t e that parental male dace i n t e r a c t i n g frequently with other dace remain at and defend t h e i r nest s i t e s longer than parental males not experiencing such i n t e r a c t i o n . The data also show that parental males take longer to respond aggressively to intruding males when they have not seen or inter a c t e d with other f i s h f o r several hours. Therefore, i t seems reasonable to conclude that i n t e r a c t i n g parental males ( i . e . , c l o s e l y spaced males) would provide greater protection f o r t h e i r nest s i t e s than more i s o l a t e d males. Parental males i n t e r a c t i n g with other males also perform considerably more substrate probing than those males without i n t e r a c t i o n . The s i g n i f i -cance of th i s difference i n substrate probing a c t i v i t y i s discussed a f t e r a l a t e r set of experiments. 72 95 35 30 -* 25! o 20 £ 15i o 10r O h 1 2 3 4 5 Interaction F i g . 19. Latency to attack by parental males for each of f i v e successive i n t e r a c t i o n s with intruding c o n s p e c i f i c males. Points represent means while v e r t i c a l l i n e s represent range. Sample s i z e f o r each of the f i v e successive i n t e r -actions i s 24. Replicates I-III are combined. 73 g. The Role of Food i n the D i s t r i b u t i o n of Dace T e r r i t o r i e s i . Outline of Experiment In order to determine whether the presence of food plays a s i g n i f i -cant r o l e i n the s e l e c t i o n and p o s i t i o n i n g of dace t e r r i t o r i e s , an experimental environment was created i n which food was a v a i l a b l e at some places but not at others. A s a t i s f a c t o r y method was devised which allowed tubifex worms to be confined to p a r t i c u l a r bottom areas of the experimental channel. Tubifex worms located i n such a manner simulated the natural prey of longnose dace, which are aquatic insect larvae and nymphs found on, under, and between stones. The following experiment created a competitive s i t u a t i o n i n which only two food areas were a v a i l a b l e to four dace. As before, l e v e l s of ag o n i s t i c a c t i v i t y were assumed to be a v a l i d i n d i c a t o r of s i t e preference. i i . Procedure The following procedure was repeated for each of si x r e p l i c a t e s . Five p e t r i dishes (11 cm diameter, 1.5 cm deep) were uniformly spaced on the channel bottom and f i l l e d with a layer of coarse gravel (3-5 cm diam). Surrounding bottom areas were covered with a layer of s i m i l a r l y sized gravel. A BC structure with both v e r t i c a l and h o r i z o n t a l walls (see F i g . 7) was placed over each p e t r i dish. Water l e v e l i n the tank was adjusted to 15.2 cm and then tubifex worms (3 ml volume) were introduced into two of the f i v e p l a t e s . Worms were added by means of a large-bore eyedropper. Thev were squirted into crevices between pieces of substrate and sank to the bottom of the plate entangling into small clumps. A f t e r 15 min water pumps were engaged. Preliminary tests showed that although worms moved about on 74 the bottom of the p e t r i p l a t e , the v e r t i c a l edges r e s t r i c t e d d i s p e r s a l to the area of the plate i t s e l f . Both the layer of rocks i n the plate and the v e r t i c a l and h o r i z o n t a l walls of the overhead structure created an area of low water v e l o c i t y . Consequently, worms i n the p l a t e were not subjected to displacement as a r e s u l t of currents. Fish fed upon tubifex by i n s e r t i n g t h e i r long snouts into crevices between rocks i n the p e t r i plate and making rapid thrusts at worms. This a c t i v i t y occasionally displaced small quantities of worms which moved downstream with the current. Such d r i f t passed through the downstream screen and out of the experimental channel. Once reaching the deeper and slower flowing elbow portions of the stream tank such worms sank to the bottom and were not r e c i r c u l a t e d . In each r e p l i c a t e , four f i s h (two male, two female) were placed i n the channel. Following a 12 hr adjustment period, 30 min observations were made at 2 hr i n t e r v a l s over a 14-1/2 hr period. Four observations were made at night and four during the day. Replicates were of short duration since continued observation might have been misleading i n the face of a diminishing food supply. Preliminary work showed that replenishing food sources without disengaging pumps and creating considerable disturbance was impossible. In most r e p l i c a t e s 30-40% of the worms remained i n the food plates at the completion of the t e s t . Number of agonistic acts won and courtship acts performed (as defined e a r l i e r ) were recorded to enable s i t e preferences and sexual motivation to be gauged. Number of feeding acts, defined as the number of successful feeding movements, were also recorded. Feeding movements involved an o r i e n t a t i o n of the body at about 45° to the h o r i z o n t a l with the long snout inserted into narrow crevices between pieces of substrate; rapid thrusts i n the d i r e c t i o n of the bottom; and a b i t i n g movement as the snout neared the bottom. Feeding movements were recorded only when worms were ingested. A f t e r each r e p l i c a t e , pumps were disengaged and p e t r i p l a t e s , gravel, and rocks were removed. Positions of the p e t r i plates containing worms were varied for each r e p l i c a t e . i i i . Experimental F i s h Twenty-four dace (twelve male, twelve female) measuring 85 to 107 mm i n fork length were used. A l l f i s h were held for 1-3 days before t e s t i n g i n tanks with tubifex worms located on the bottom. i v . Results and Discussion The data from a l l r e p l i c a t e s were combined and analyzed with the Wilcoxon matched-pairs signed-ranks t e s t . Each dace was compared against i t s e l f i n performing the matched pai r s t e s t s . Since there were more empty areas than food areas, s t a t i s t i c a l comparisons were made on a per area b a s i s . The r e s u l t s show that although male dace fed from the p e t r i plates containing tubifex worms, they performed a s i g n i f i c a n t l y greater number of defences at empty plates (p<.025) than at those with worms (Fig. 20). In agreement with p r i o r r e s u l t s (Fig. 8), a s i g n i f i c a n t l y greater proportion of male agonistic a c t i v i t y (p<.025) occurred at night (Fig. 20). During night hours, males courted females (Fig. 20) and frequently allowed females 76 F i g . 20. Mean number of a g o n i s t i c , courtship, and feeding acts performed per food and no food area by male and female dace during day and night. Replicates I-VI are combined. Twenty-four dace (twelve male, twelve female) were tested. (Agonistic and courtship acts occurred at areas free of structures also, see text) 76a Ma les B agonistic (won) D courtship E3 feeding FOOD NO FOOD Females 55 1 FOOD 1 NO FOOD 77 to enter t h e i r t e r r i t o r i e s , even when the holding was over a food area. Females, however, showed a s i g n i f i c a n t l y greater number of defences at p e t r i plates containing food (p = .01) than at empty ones ( F i g . 20). Most of these, defences (p<.005) were during the day (Fig. 20) and were performed almost equally (p>.05) against males and females (Table V I I I ) . A female was observed to feed outside the area she defended only once, whereas a male was observed to leave i t s t e r r i t o r y to feed elsewhere on f i v e separate occasions. One r e p l i c a t e had to be repeated since three spawning acts occurred during the t e s t . However, i t i s i n t e r e s t i n g to note that the spawnings took place over an empty p e t r i p l a t e . In addition to defending the areas over p e t r i p l a t e s , male dace also defended areas free of enclosures. Although adequate structures of the BC type were a v a i l a b l e (5 structures: 4 f i s h ) , males nevertheless defended these "open" areas 62 times. These defences represented 26.7% of the t o t a l number of a g o n i s t i c acts won by males. Of these a g o n i s t i c acts won over open areas, 79% were at night and a l l night defences were against other males. Females never defended such open areas. Since open areas consisted of coarse gravel, t h i s information supports e a r l i e r findings (Fig. 8) which showed that male t e r r i t o r i a l i t y was r e s t r i c t e d to areas with coarse bottom substrate. As i n e a r l i e r experiments (see Table IV), females displayed better success during a g o n i s t i c i n t e r a c t i o n s with males, winning 76.7% compared with 23.3% for males (Table V I I I ) . Once again, however, t h i s d i f f e r e n c e i s a t t r i b u t a b l e to the f a c t that females were more f a i t h f u l to t h e i r occupied areas, and thus were usually residents.being intruded upon by males. 78 Table VIII. Mean number of ago n i s t i c acts won per food and no food area by male and female dace against the same and opposite sex during day and night. Numbers i n parentheses i n d i c a t e t o t a l number of wins. Replicates I-VI are combined. Twenty-four dace (twelve male, twelve female) were tested. Mean No. Agonistic Acts Food No Food Combined won/area Day Night Day Night Day Night by female against female 29.0 2.5 5.0 8.3 14.6 6.0 (73) (30) by female against male 26.0 7.5 15.0 6.7 19.4 7.0 (97) (35) by female.against both female and male 55.0 10.0 20.0 15.0 34.0 13.0 (170) (65) by male against male 6.5 10.0 12.7 19.7 10.2 15.8 (51) (79) by male against female 5.0 3.0 3.3 4.7 4.0 4.0 (20) (20) by male against both male and female 11.5 13.0 16.0 24.3 14.2 19.8 (71) (99) 79 The r e s u l t s i n d i c a t e that the presence of food i s not necessary f o r the expression of t e r r i t o r i a l i t y by e i t h e r male or female dace. However, females did s e l e c t and defend shelters containing food and thus the presence of food may be a proximate factor involved i n the s e l e c t i o n of t e r r i t o r i e s by females. For males, evidence instead points to the coarse bottom substrate as the f a c t o r determining where they s e l e c t t e r r i t o r i e s . . The b r i e f experi-ments reported on here leave unresolved the question of whether food p r o v i -sion may be an ultimate factor i n male and/or female t e r r i t o r y . What hope-f u l l y has been demonstrated i s the l i k e l i h o o d that, at l e a s t for female dace, the d i s t r i b u t i o n of food appears to influence the s e l e c t i o n of t e r r i -tory s i t e s . Although food i s abundant and r e l a t i v e l y uniformly dispersed throughout the r i f f l e h a b i t a t , i t seems p l a u s i b l e that s p e c i f i c areas may be e s p e c i a l l y a t t r a c t i v e to dace i f d r i f t patterns c o n t i n u a l l y r e p l e n i s h food supplies there. The r e s u l t s of the foregoing experiment harbour an uncertainty. The competitive nature of the t e s t s (two food areas: four f i s h ) coupled with the 'dominance' of females over males may create a somewhat misleading s i t u a t i o n with respect to male preferences for food and no food areas. To remedy th i s problem i t was resolved to examine males and females separately. However, attempts to test females alone (three food areas: three no food areas: three females) f a i l e d to provide any a d d i t i o n a l information on the r o l e of food i n female t e r r i t o r y . Female t e r r i t o r i a l behaviour was sporadic and the measures of a g o n i s t i c acts won, used to i n d i c a t e s e l e c t i o n of t e r r i t o r i e s , ' were not attainable. These r e s u l t s were unexpected and a c t u a l l y prompted the e a r l i e r reported experiments on s o c i a l factors a f f e c t i n g 80 t e r r i t o r i a l behaviour i n female R. c_. d u l c i s (page 45 ) . Nine male dace (89 to 105 mm i n fork length) tested alone i n three r e p l i c a t e s (three food areas: three no food areas: three males) behaved s i m i l a r l y to when tested with females (compare F i g . 20 and F i g . 21). P o s i t i o n checks made at 15 min i n t e r v a l s during the 30 min observations showed that males occurred more frequently at no food areas than at food areas (Fig. 21A). During both.day and night, these males defended no food areas s i g n i f i c a n t l y more (p<.01) than, food areas (Fig. 21B) . Although vacant shelters were a v a i l a b l e , dace often defended open areas at night (Fig. 21B) . This agrees with previous r e s u l t s and once again stresses the importance of the coarse spawning gravel component i n the s e l e c t i o n of territories"byiamale<adace. B. Non-Breeding Phase a. S i t e Preferences of Male and Female Dace i n Late Summer and Winter i . Outline of Experiment Outside the breeding phase, longnose dace t e r r i t o r i a l i t y i s consider-ably relaxed. This alone suggests that longnose t e r r i t o r i e s are r e l a t e d p r i m a r i l y to reproductive a c t i v i t i e s . The requirements of dace c e r t a i n l y d i f f e r from breeding to non-breeding phases, and i t i s to be expected that these differences w i l l be r e f l e c t e d i n the s o c i a l structure. For compara-t i v e purposes, experiments on s i t e preferences conducted during the spring with breeding dace were repeated during the l a t e summer (September) and winter (January) of the non-breeding phase. 81 c 4) 5 0 30 o 4 0 C o 5 30 c o O) < 2 0 h 10 • day B night FOOD NO FOOD OPEN F i g . 21. Percent occurrence (A) and t o t a l number of ago n i s t i c acts won (B) at food, no food, and open areas by male dace during day and night. Replicates I-III are combined. A t o t a l of nine males were tested. 82 i i . Procedure The experiment conducted to determine what factors are involved i n the s e l e c t i o n of a t e r r i t o r y by dace was repeated with l a t e summer and winter c o l l e c t e d f i s h . Four recordings were made under both day and night conditions. However, since agonistic acts by non-breeding dace were infrequent, p o s i t i o n checks were also made at 15 min i n t e r v a l s during the 30 min recording periods. I f dace.were i n motion during a p o s i t i o n check t h e i r p o s i t i o n was recorded once they remained stationary f o r 10 sec or more. In addition to the s i x types of structures described e a r l i e r (see F i g . 7), the p o s i t i o n r e f e r r e d to as "open" and abbreviated "0" was used to designate dace not positioned at any structure. Only two instead of three winter r e p l i c a t e s were performed, since i t i s exceedingly d i f f i c u l t to c o l l e c t large numbers of dace at t h i s time. i i i . Experimental F i s h Eighteen dace (nine male, nine female), measuring 82 to 105 mm i n fork length, were used f or l a t e summer r e p l i c a t e s and twelve (s i x male, s i x female), measuring 80 to 117 mm i n fork.length, were used f or winter r e p l i c a t e s . i v . Results and Discussion Unlike breeding i n d i v i d u a l s , dace c o l l e c t e d i n l a t e summer displayed l i t t l e a gonistic behaviour (Table IX). In addition to quan t i t a t i v e d i f f e r -ences, q u a l i t a t i v e differences also existed between the agonistic behaviour performed by dace during breeding and non-breeding phases. When ago n i s t i c a c t i v i t y occurred during the non-breeding phase, i t was i n the form of the Table IX.. Mean number of agonistic acts won per area per 30 min by male and female dace during l a t e summer. Replicates I-III are combined. Eighteen dace (nine male, nine female) were tested. Mean Number of Agonistic Acts Won/area/30 min BC BF CC CF SC SF 0 Time of Day Female Male Female Male Female Male Female Male Female Male Female Male Female Male Day 0.16 0 0.07 0 0.13 0.05 0 0 0 0 0 0 0 0 Night 0.02 0 0 0 0 0 0 0 0 0 0 0 0 0 Day and Night 0.18 0 0.07 0 0.13 0.05 0 0 0 0 0 0 0 0 84 weaker behaviour patterns of 'butting' and ' b i t i n g ' . The a g o n i s t i c behaviour patterns of 'darting', 'chasing', and ' f i g h t i n g ' , however, were not common outside the breeding phase (Table X). Table X. Agonistic behaviour patterns commonly performed (X) by breeding and non-breeding dace. Behaviour Pattern Breeding Non-Breeding Butt X X B i t e X X Dart X -Chase X -Fight X — Figure 22 shows a s t r i k i n g s i m i l a r i t y i n male and female s i t e preferences during l a t e summer. Females were not r e s t r i c t e d to BC and BF areas, and males no longer showed r i g i d preferences for coarse bottom substrate at night (compare F i g . 22 with F i g . 8). Both sexes occupied BF structures most frequently (33 - 54.6%) during both day and night and more than 80% of a l l p o s i t i o n checks found two or more dace (of the same or both sexes) sharing such structures. BC and BF structures together were the preferred s i t e s accounting for 76.4% occurrence of females and 64.2% occurrence of males during the day and 51.1% occurrence of females and 38.8% occurrence of males at night. Marked behavioural differences 0 85 45 30 o » < 15 « a c « 15 30 BC IF BF DAY D females B males CF SC I f NIGHT F i g . 22. Percent occurrence of male and female dace at d i f f e r e n t areas during l a t e summer. Only frequencies larger than 1% are shown. Replicates I-III are combined. Eighteen dace (nine male, nine female) were tested. 86 between male and female dace are therefore r e s t r i c t e d to the breeding phase. Vertebrate populations commonly show seasonal changes i n s o c i a l organization involving a l t e r a t i o n s i n the r e l a t i o n s between the sexes. The changing s o c i a l grouping of fishes during reproductive a c t i v i t i e s i s complex and varies widely with the species involved (Breder, 1959). Fishelson (1970) has shown that the sergeant major f i s h change from feeding schools of mature f i s h to breeding colonies of males that court females which remain i n schools. Out of the breeding season both sexes of three-spined stickleback foarm aggregations or schools, but when the males develop t h e i r breeding colors they become aggressive and disperse. Females con-tinue to school u n t i l egg laying begins. Dace c o l l e c t e d during winter displayed no observable a g o n i s t i c behaviour. Dace were l e t h a r g i c and p o s i t i o n changes which were common i n spring and l a t e summer were infrequent under winter conditions. Unlike s i t e preferences of dace during.spring and l a t e summer periods, a l l winter dace r e s t r i c t e d themselves to enclosures (BC and BF) (Fig. 23). Occasional movements outside the enclosures accounted for the occurrence at open areas. Although food was a v a i l a b l e throughout the channel, dace fed infrequently. Reduction i n metabolic rate and a c t i v i t y are general phenomena of p o i k i l o -therms which account for the reduced food intake. Densities at enclosures were high and dace were often pressed f i r m l y against other f i s h . Under these conditions, dace were able to remain stationary without any movement of the f i n s or body. During winter, f i e l d dace are generally located under large rocks several layers deep i n the substrate. Such behaviour may prevent downstream DAY G females B males BC BF CC CF SC" SF NIGHT F i g . 23. Percent occurrence of male and female dace at d i f f e r e n t areas during winter. Replicates I and II are combined. Twelve dace ( s i x male, s i x female) were tested. 88 displacement and ice-scouring during the winter period when highest discharges occur i n the r i v e r . The chances of predation by the winter run of adult steelhead trout and salmon would also be reduced by such hiding behaviour. Edmundson et a l . , (1968) found that winter locations of young chinook salmon and steelhead trout were p r i m a r i l y under or between rubble p a r t i c l e s . Hartman (1965) reported that j u v e n i l e steelhead trout hide during winter while j u v e n i l e coho salmon form dense groups. He suggested that these responses protected the f i s h against predation, downstream displacement, and ice-scouring. b. Seasonal V a r i a t i o n i n the Spacing Patterns of Dace i . Outline of Experiment The preceding section has shown that s i t e preferences of male and female dace change s i g n i f i c a n t l y from season to season. I t also suggests that the grouping tendencies or spacing patterns of dace change markedly as w e l l . The objective of the following experiment was to assess the seasonal changes i n spacing patterns of adult dace, and to determine what rol e the r e l a t i o n s between i n d i v i d u a l s ( i . e . , i n t e r a c t i o n s ) played i n the observed spacing patterns. Spacing patterns of spring, l a t e summer, and winter c o l l e c t e d dace were compared by pl a c i n g a constant number of dace in t o a standardized experimental r i f f l e . A gonistic i n t e r a c t i o n s between dace were recorded and the grouping conditions under which they occurred were noted. i i . Procedure Two 3-day r e p l i c a t e s were performed with dace c o l l e c t e d during the 89 spring (breeding phase), l a t e summer, and winter. The standardized r i f f l e i n a l l tests consisted of s i x enclosures (BC) uniformly spaced on the experimental channel bottom. A l l remaining bottom areas were covered with coarse gravel. In each r e p l i c a t e , s i x dace (three male, three female) c o l l e c t e d during a p a r t i c u l a r season were placed i n the channel. A f t e r a 12 hr adjustment period, four 30 min recordings were made during both day and night. P o s i t i o n checks of i n d i v i d u a l dace were made at 15 min i n t e r v a l s . Number of a g o n i s t i c acts performed by s o l i t a r y dace and dace i n groups were scored. i i i i . Experimental Fish Spring (May) c o l l e c t e d dace ( s i x male, s i x female) ranged from 85 to 106 mm i n fork length, l a t e summer (September) c o l l e c t e d dace ( s i x male, si x female), from 87 to 103 mm i n fork length, and winter (January) c o l -l e c t e d dace ( s i x male, s i x female), from 77 to 107 mm i n fork length. i v . Results and Discussion The data on spacing patterns of dace were compared with Contingency Chi-square t e s t s . Frequency of occurrence of d i f f e r e n t group sizes were calculated as shown e a r l i e r (see page 46). Spacing patterns of dace ( i . e . , occurrence of group sizes.oone, two, and three or more) i n the standardized experimental r i f f l e changed s i g n i f i -cantly from spring to l a t e summer (p<. 001) and from l a t e summer to winter (p<.001; F i g . 24). Breeding dace were characterized by a predominance of s o l i t a r y i n d i v i d u a l s (both male and female), a small percentage of p a i r s , and progressively fewer groups of three and four. Mean group s i z e f o r t h i s phase was only s l i g h t l y more than one ( F i g . 25A). Outside the breeding 90 1 2 3 4 4 + GROUP SIZE F i g . 24. Percent of occurrence of d i f f e r e n t group sizes of dace during spring, l a t e summer, and winter seasons. Replicates I and II are combined. Twelve dace ( s i x male, s i x female) were tested for each season. 91 F i g . 25A. Relationship between t o t a l number of agonistic acts per-formed and mean group sizes of dace during spring, l a t e summer, and winter seasons. Replicates I and II are combined. Twelve dace ( s i x male, s i x female) were tested, f o r each season. F i g . 25B. Relationship between t o t a l number of agonistic acts per-formed, number of ago n i s t i c acts won ( i n parentheses), and d i f f e r e n t group sizes of dace during spring, l a t e summer, and winter seasons. Replicates I and II are combined. Twelve dace ( s i x male, s i x female) were tested f o r each season. 91a A . 200 • < y IOO z o o < © Spring O Late Summer •Winter oh -A MEAN GROUP SIZE J I 1 I l _ 1 2 3 4 4+ GROUP SIZE phase, numbers of single dace occupying enclosures decreased while groups of two, three, four, and four plus became more common ( F i g . 24). Mean group s i z e during the l a t e summer exceeded two (Fig. 25A) . During winter, clumps or l a r g e r group sizes were most prominent (Fig. 24) as mean group s i z e approached three. The data on i n t e r a c t i o n s between dace suggest that a g o n i s t i c beha-viour i s the mechanism promoting the observed spacing pattern of breeding dace. S o l i t a r y dace account f o r the majority -of agonistic acts performed during the spring breeding season, while those dace i n pa i r s and i n groups of three or four show progressively l e s s a g o n i s t i c behaviour (Fig. 25B). Frequency of success during agonistic i n t e r a c t i o n s ( i . e . , a g o n i s t i c acts won) follows a s i m i l a r pattern f o r each group s i z e ( F i g . 25B). S o l i t a r y dace are almost always successful i n defending t h e i r t e r r i t o r i e s against intruders, but a g o n i s t i c behaviour performed by dace i n l a r g e r groupings often passes with no change i n group s i z e occurring. During l a t e summer and winter seasons, agonistic behaviour occurs infrequently ( Fig. 25). Therefore, i t is.suggested that t h i s d i f f e r e n c e i n the amount of agonistic behaviour performed by breeding and non-breeding dace accounts f o r the establishment and maintenance of d i f f e r e n t group s i z e s . In the experimental channel, winter dace often crowded into one of the a v a i l a b l e shelters ( i . e . , more than four dace at one enclosure). In l a t e summer, sh e l t e r s i t e s s i m i l a r l y formed l o c i f o r the formation of aggregations. Unlike winter groupings, however, such l a t e summer groups were continually changing i n s i z e and l o c a t i o n . 93 Seasonal changes i n spacing patterns of dace correspond with f l u c t u a t i o n s i n water flow. Such f l u c t u a t i o n s i n flow ult i m a t e l y deter-mine the amount of r i f f l e habitat available to dace. In the spring, when dace space out by claiming i n d i v i d u a l t e r r i t o r i e s , flows are high and r i f f l e areas are p l e n t i f u l . During l a t e summer, water flows diminish and dace are concentrated i n t o r i f f l e habitats of reduced area. At th i s time of year dace are able to remain close to one another without consider-able antagonism. c. Role of Food i n the D i s t r i b u t i o n of Dace i n Late Summer i . Outline of Experiment Food l o c a l i z a t i o n experiments i d e n t i c a l with those conducted with breeding dace were repeated with dace c o l l e c t e d during the l a t e summer. Tests were made to determine what role the presence of food might play i n the d i s t r i b u t i o n of dace within r i f f l e s during the non-breeding phase. i i . Procedure Procedures were e s s e n t i a l l y i d e n t i c a l to those already described, except f o r appropriate temperature and photoperiod changes. Once again, however, p o s i t i o n checks were made (three per 30 min) and calculated per-cent occurrences were assumed to be i n d i c a t i v e of s i t e preference. As i n previous experiments the "open" p o s i t i o n was used f o r recording pur-poses. Feeding acts as defined e a r l i e r were also recorded. i i i . Experimental F i s h Twenty-four dace (twelve male, twelve female) measuring 80 to 101 mm i n fork length were used. 94 i v . Results and Discussion -Agonistic acts, although infrequent, were won at both food and no food areas (Table XI). Both males and females fed from p e t r i plates containing tubifex worms (Table XI), but occurrence at food areas was not s i g n i f i c a n t f o r e i t h e r males (p>.05; Wilcoxon matched p a i r s test) or females (p>.05; F i g . 26). Once again each f i s h was compared against i t s e l f i n performing the matched pai r s t e s t s . Two or more dace (same or both sexes) were found sharing shelters over.food plates on 30% of the p o s i t i o n checks, while 51% of the checks recorded t h i s s i t u a t i o n at no food areas. During the e a r l i e r breeding phase food experiments, male dace but never females were observed, to defend areas free of any structures. The present experiment found both sexes to occur frequently at these open areas, p a r t i c u l a r l y at night ( F i g . 26). During observations, dace of both sexes often swam throughout the experimental channel, t e s t i n g the substrate f o r the presence of food. The re s u l t s suggest that i t i s u n l i k e l y that food determines the d i s t r i b u t i o n of dace during the non-breeding phase. Within the r i f f l e environment food i s generally uniformly abundant and accessible by forays from p o s i -tions of cover and s h e l t e r . C. Spacing Patterns of Dace Fry i . Outline of Experiment To observe movements and spacing patterns of dace f r y , eggs spawned i n the stream tank were l e f t to hatch. Fry were allowed to grow to 20 to 30 mm i n length. 95 Table XI. Mean number of agonistic acts won and feeding acts performed by dace per food, no food, and open area during l a t e summer. Replicates I-VI are combined. Twenty-four dace (12 male, 12 female) were tested. Mean number of acts/area Behavioural Act Food female male No female Food male Open female male Combined female male Agonistic - won (day) 4.5 0.5 2.7 2.7 0 0 3.4 1.8 Agonistic - won (night) 0 0 0 0 0 0 0 0 Agonistic - won (day and night) 4.5 0.5 2.7 2.7 0 0 3.4 1.8 Feeding (day) 6.0 1.5 0 0 0 0 2.4 0.6 Feeding (night) 0 0.5 0 0 0 0 0 0.2 Feeding (day and night) 6.0 2.0 0 0 0 0 2.4 0.8 96 50 30 at < UJ a. U J U Z UJ O f U O 10h 10 30 50 FOOD NO FOOD DAY • fe males @ males OPEN NIGHT F i g . 26. Mean percent occurrence of male and female dace per food, no food, and open area during l a t e summer. Replicates I-VI are combined. Twenty-four dace (twelve male, twelve female) were tested. 97 i i . Procedure A f t e r eggs were spawned i n the experimental r i f f l e areas of the stream tank, upstream and downstream screens were removed and sloping f l o o r s werers added to each end of the f a l s e bottoms. In t h i s manner, an a l t e r -ating r i f f l e - p o o l environment was created i n which the elbows of the tank provided the deeper and slower flowing pool zones. Several rocks and a layer of gravel were placed on the bottom of the pool zones. Once eggs hatched, f r y were observed at i r r e g u l a r l y spaced i n t e r v a l s f o r several weeks. i i i . Results and Discussion Fry were f i r s t noticed i n the pool areas 16 days a f t e r eggs were spawned. At t h i s time, f r y were approximately 10 to 20 mm i n length and formed large loose aggregations near the water surface. Not a l l i n d i v i -duals i n such aggregations were uniformly spaced nor oriented i n the same d i r e c t i o n . The majority of f r y within any p a r t i c u l a r grouping, however, were spaced one to three f i s h lengths apart ( i . e . , 10 to 30 mm) and were oriented i n approximately the same d i r e c t i o n . Aggregations of dace f r y observed i n the f i e l d were s i m i l a r l y spaced. Such aggregations of a hundred or more f r y were not uncommon. As f r y i n the stream tank grew, they appeared i n smaller groups (less than 12) near the bottom i n the pool zone. Occasionally i n d i v i d u a l f r y were observed foraging alone. Once dace reached 20-30 mm i n length, they began moving over the f a s t flowing r i f f l e areas and maintained p o s i -t i o n over the bottom. Gibbons and Gee (1972) reported that young of the year longnose dace move in t o fast water i n J u l y and August when they are 98 between 25 and 30 mm i n length. F i e l d c o l l e c t i o n s made i n the present study produced s i m i l a r f i n d i n g s . Dace f r y i n the experimental r i f f l e areas displayed a preference for areas with overhead cover, adjusting t h e i r positions i n response to experimental r e l o c a t i o n of t h i s environmental feature. Since adult dace show t h i s same association with overhead cover, e s p e c i a l l y during the day, i t seems possible that overhead objects serve as a v i s u a l cue upon which dace f i x . The somewhat d o r s a l l y located eyes of longnose dace would be w e l l adapted i n t h i s case. The agonistic behaviour patterns of darting and b i t i n g were observed among several c l o s e l y spaced f r y i n the r i f f l e area. Although speculative, i t seemed that agonistic a c t i v i t y i n these small groups served to disperse f r y throughout the r i f f l e , p a r t i c u l a r l y upstream. Fry fed almost exclusive-l y on the algae encrusted rocks. F i e l d observations of j u v e n i l e dace, 25 to 45 mm i n length, made by Gibbons (MS, 1971) found these f i s h u sually not more than 1 cm off the bottom and i n groups of three to ten. Spaces occupied by y e a r l i n g dace are small. Laboratory observations indi c a t e that these younger f i s h do not enter into competition with older dace u n t i l they a t t a i n a larger s i z e and require larger spaces. Habitat requirements of A t l a n t i c salmon are also known to change as the f i s h grow (McCrimmon, 1954). Hynes (1970) suggests that a s i m i l a r pattern of habitat adjustment occurs i n aquatic insect nymphs i n rapid water environments where she l t e r i s c r i t i c a l and growing nymphs require i n c r e a s i n g l y large crevices. 99 D. Discussion of the Causation of T e r r i t o r i a l Behaviour The data on t e r r i t o r y s e l e c t i o n by longnose dace, Rhinichthys £. d u l c i s , demonstrate a sharp contrast i n the behaviour of the sexes. Males are most t e r r i t o r i a l at night, defending coarse substrate areas against other males. Male courtship i s also nocturnal and occurs over these same areas. During the day, however, reproductive a c t i v i t y i s relaxed, and males no longer show a r i g i d preference f o r coarse substrate areas. Therefore i t i s concluded that the major factor involved i n the s e l e c t i o n of t e r r i t o r i e s by male dace i s coarse spawning substrate. Females, unlike males, are not more t e r r i t o r i a l at night. They do not e x h i b i t any bottom substrate preferences, but defend only enclosed areas. Once receptive, females leave preferred areas and court and spawn with t e r r i t o r i a l males at preferred areas f o r courtship and spawning. These l a t t e r areas are preferred for both defence and courtship by males. Females s e l e c t and defend shelters containing food and thus the presence of food may be a proximate factor involved i n the s e l e c t i o n of t e r r i t o r i e s by female dace. Although females feed on the t e r r i t o r y t h i s does not n e c e s s a r i l y show that the presence of food i s a s i g n i f i c a n t and advantageous consequence of female t e r r i t o r y , unless proximate and ultimate factors correspond. S i m i l a r l y , c o l l e c t i o n of food outside the t e r r i t o r y by males i s not proof that male t e r r i t o r y has no s i g n i f i c a n c e i n r e l a t i o n to food. In the r i f f l e environment i t may be that once a s u i t a b l e s i t e i s secured,food sources w i t h i n the immediate v i c i n i t y can be harvested. For example, dace might base the s e l e c t i o n of a t e r r i t o r y on the type of sub-s t r a t e and/or v e l o c i t y i n an area (proximate f a c t o r s ) , but the abundance 100 of food found i n the area might provide the s e l e c t i v e advantage f o r making t h i s choice and thus be the ultimate factor involved. Habitat s e l e c t i o n tests by Sale (1969) with j u v e n i l e surgeon f i s h and by Baker (1971) with four-spined sticklebacks have led them to agree with Hilden's (1965) conclusions that s i t e s e l e c t i o n i s not l i k e l y to be a response d i r e c t l y to ultimate f a c t o r s , but to a s e r i e s of proximate f a c -tors. Thus by responding to a ser i e s of proximate factors the ultimate factors are acquired. Circumstances i n which an adequate food source i s a v a i l a b l e within the confines of the t e r r i t o r y may be preferable f o r female dace. Under these circumstances,frequent feeding movements outside the t e r r i t o r y possibly i n v o l v i n g encounters and c o n f l i c t s with males would not be r e -quired. The amount of feeding that may be done within the t e r r i t o r y i s c e r t a i n l y not inconsiderable and may be important during the b r i e f period p r i o r to ovulation when food intake i s markedly increased (Bartnik, pers. observation). Symons (1968) hypothesized that i f the function of t e r r i t o r i a l be-haviour of some species of f i s h i s food r e l a t e d , the s i z e of the t e r r i t o r i e s should increase when food i s scarce and decrease when food i s abundant. Changes i n t e r r i t o r y s i z e would be brought about by appropriate increases and decreases i n aggression. Such increased aggression upon deprivation, of food has been observed i n A t l a n t i c salmon (Symons, 1968), i n coho salmon (Mason and Chapman, 1965), and i n medaka (Magnuson, 1962). Dace held i n the stream tank and deprived of food for 4-1/2 days f a i l e d to show any s i g n i f i c a n t increase i n aggressive behaviour. 101 E a r l i e r experiments (see pages 45-55 ) indicated that i n t e r a c t i o n with male dace i s a causative agent for the expression of t e r r i t o r i a l behaviour i n female dace. I t i s therefore argued that the difference i n s o c i a l structure ( i . e . , female t e r r i t o r i a l i t y ) between the two populations of dace described here i s a t t r i b u t a b l e to the b i o t i c or s o c i a l factors of either segregation or coexistence of the sexes. Where the sexes coexist, the phenomenon of female t e r r i t o r i a l i t y apparently serves to reduce con-f l i c t s between males and females. Male dace i n breeding condition are attracted to and i n t e r a c t with other males. This behaviour r e s u l t s i n the formation of t e r r i t o r y c l u s t e r s . The fact that some t e r r i t o r i a l song b i r d s seem to "clump together" at low population d e n s i t i e s has been suggested as evidence that d i s p l a y i n g to neighbours has a'reward' value (pers. comm., J.R. Krebs). Perhaps a si m i -l a r reason e x i s t s f o r the observed c l u s t e r i n g of male dace t e r r i t o r i e s . The importance of s o c i a l i n t e r a c t i o n f o r the expression of t e r r i t o r i a l behaviour and the formation.of t e r r i t o r i a l groupings has been reported for various f i s h species. Van den Assem (1967) found that under c e r t a i n condi-t i o n s , s e t t l i n g and nest-building of three-spined sticklebacks i s activated by i n t e r a c t i o n with c o n s p e c i f i c s . Abel (1961) and Myrberg et a l . , (1967) have emphasized that group nesting by pomacentrid f i s h seems to r e s u l t from s o c i a l responses. Keenleyside (1970) has described male sunfish as being strongly drawn towards each other when es t a b l i s h i n g breeding t e r r i t o r i e s . In the behavioural l i t e r a t u r e , the term habituation i s often used to describe the reduction of aggressive i n t e r a c t i o n between t e r r i t o r i a l neighbours. Several recent papers have demonstrated that the aggressive 102 responses of t e r r i t o r i a l f i s h w i l l habituate when the f i s h are exposed repeatedly to conspecifics confined to glass tubes (Peeke, 1969; Peeke and Peeke, 1970), free-swimming adjacently t e r r i t o r i a l neighbours (van den Assem and van der Molen, 1969; Peeke et a l . , 1971; Gallagher et a l . , 1972), or crude models of conspecifics (Peeke et a l . , 1969; Peeke, 1969). In the present study, however, the aggressive behaviour of i n t e r a c t i n g clustered t e r r i t o r i a l male dace did not habituate. Instead, such males continued to attack intruders from neighbouring t e r r i t o r i e s . Although these findings appeared.to be at odds with the l i t e r a t u r e c i t e d above, they are more tenable i n the l i g h t of the most recent i n v e s t i g a t i o n s (Peeke and Veno, 1973) of habituation. In t h e i r laboratory t e s t s , Peeke and Veno c l e a r l y demonstrated that t e r r i t o r i a l f i s h can discriminate i n d i v i d u a l morphological cues as w e l l as geographic p o s i t i o n cues. This led them to conclude that widespread g e n e r a l i -zation of habituation does not occur. These findings a c t u a l l y confirmed the b e l i e f held by the authors of the e a r l i e r habituation studies that both the v a r i a b i l i t y of the r e l e a s i n g s t i m u l i and the occurrence of i n t e r -mittent consummatory acts ( i . e . , reinforcement through v i c t o r y ) i n nature would protect f i s h against habituation. Outside the breeding phase, the cues used by dace i n s i t e s e l e c t i o n change markedly and male and female dace d i f f e r l i t t l e i n the areas they choose to occupy. Agonistic behaviour i s infrequent among non-breeding dace and as a r e s u l t the d i s t r i b u t i o n of dace wit h i n r i f f l e s becomes con-tagious or clumped, p a r t i c u l a r l y during winter. 103 During the non-breeding phase ( i . e . , l a t e summer) shelters serve as l o c i f o r the formation of aggregations of dace. However, at t h i s time neither sex appears to respond to the presence of food i n s e l e c t i n g p a r t i c u l a r s i t e s . Many b i r d species form t e r r i t o r i e s i n the breeding season but occur i n flo c k s at other times of the year, and these changes co r r e l a t e with a change i n food dispersion from an evenly dispersed supply to one that i s patchy i n d i s t r i b u t i o n (Crook and Goss-Custard, 1972). Changes i n spacing patterns of longnose dace from breeding to non-breeding seasons, however, appear unrelated to food dispersion i n r i f f l e s which probably remains r e l a t i v e l y constant through spring and summer months. 104 SECTION I I I THE FUNCTIONAL SIGNIFICANCE OF THE SOCIAL ORGANIZATION OF DACE A. Introduction Hinde (1956, 1969) has pointed out that two rather d i f f e r e n t meanings of " f u n c t i o n " are sometimes confused. The term i s often used to mean any advantageous consequence. A l t e r n a t i v e l y , other authors (Tinbergen, 1957, 1965; Brown and Orians, 1970) r e s t r i c t the meaning to b i o l o g i c a l l y s i g n i -f i c a n t consequences—that i s , those through which s e l e c t i o n i n favour of the behaviour can act. Tinbergen states that to conclude that one has demonstrated or determined what the function of a c e r t a i n behaviour i s , one must experimentally demonstrate s e l e c t i o n pressures that prevent the species from deviating from i t s present state. In h i s paper "Behaviour and Natural S e l e c t i o n " , Tinbergen (1965) discusses a number of studies that have taken the necessary experimental approach i n attempting to determine the function of various behavioural t r a i t s ( i . e . , c o l o n i a l nesting, voca-l i z a t i o n s , parental fanning, e t c . ) . The comparative method has proved p a r t i c u l a r l y f e r t i l e to the study of the adaptive s i g n i f i c a n c e of s o c i a l organizations. This approach makes use of c l o s e l y r e l a t e d species which may d i f f e r s t r i k i n g l y i n t h e i r beha-viour. In extensive f i e l d studies, Crook (1965) has shown that by corre-l a t i n g e c o l o g i c a l v a r i a b l e s with s p e c i f i c population dispersion types the adaptive s i g n i f i c a n c e of s o c i a l organizations can be i n f e r r e d . Since Howard (1920) wrote his c l a s s i c book on b i r d t e r r i t o r i a l i t y , b i o l o g i s t s have sought an answer to the question: "What i s the function of 105 t e r r i t o r i a l behaviour?" Many n a t u r a l i s t s have ascribed functions to t e r r i t o r i a l i t y mainly on i n f e r e n t i a l bases, but co n t r o l l e d experimentation ( i . e . , d i r e c t evidence) i s lacki n g . Few studies of t e r r i t o r i a l behaviour have a c t u a l l y answered the question: "Would deviations from the observed norm be penalized, and i f so, how?". One of the basic problems confronting behaviourists studying function i s the f a c t that, unlike s t r u c t u r a l characters, behavioural ones are more d i f f i c u l t to i s o l a t e and experimentally manipulate. However, Tinbergen (1965) argues that p e r f e c t l y good methods are a v a i l a b l e f or the study of environmental pressures imposing demands on an animal's behaviour. Ex-perimental tests of hypotheses on various aspects of dace t e r r i t o r i a l be-haviour follow i n l a t e r sections. B. Inferences from Observations and Experiments i n Section II as to the Function of Various Aspects of T e r r i t o r i a l Behaviour The observed r e l a t i o n s h i p (see Section II) between courtship, defence, s u i t a b l e spawning substrate, and time of day leads me to i n f e r that an important function of male dace t e r r i t o r y i s the p r o v i s i o n of a s i t e i n which to court and spawn with females. Since longnose dace do not a c t i v e l y bury t h e i r eggs by moving the substrate (Bartnik, 1970), only coarse sub-s t r a t e areas which provide natural depressions are s u i t a b l e for egg deposi-t i o n . Eggs deposited on f i n e gravel would be exposed to s i l t a t i o n and mechanical damage from a s h i f t i n g substrate. In addition, necessary r e -fuges for newly hatched alevins would be lacking. A probable second function of male t e r r i t o r y i s the protection of newly deposited eggs from 106 predation. Because t e r r i t o r i a l male dace continue to defend the t e r r i t o r y a f t e r spawning, t h i s would seem to be a reasonable inference to make. The observations and experiments i n Section I I , however, lead me to make a very d i f f e r e n t inference as to the function of female t e r r i t o r y . The experiments on t e r r i t o r y s e l e c t i o n and s o c i a l factors a f f e c t i n g female t e r r i t o r i a l behaviour suggest that female t e r r i t o r y functions, at l e a s t i n part, i n maintaining a sheltered area and thereby reducing c o n f l i c t s with males. Such c o n f l i c t s ( i . e . , courtship harassment and a g o n i s t i c i n t e r -action) might cause females to be frequently displaced from s h e l t e r s . Females continually swimming about i n the strong current of the r i f f l e habitat may experience s t r e s s f u l conditions, and perhaps become vulnerable to predation. Other inferences that can be drawn from the preceding section deal with the c l u s t e r i n g of male t e r r i t o r i e s . I t seems possible that clustered t e r r i t o r i a l males experience greater reproductive success than more i s o l a t e d males. F i n a l l y , i t seems l i k e l y that dace t e r r i t o r i a l behaviour may also l i m i t the density of breeding f i s h i n a given area, and therefore act as a dispersing mechanism. Experimental tests of some of the inferences drawn on the functions of t e r r i t o r i a l behaviour are presented i n the following pages. C. Experimental Tests of Hypotheses Regarding Function a. S i g n i f i c a n c e of T e r r i t o r y Clusters i . Outline of Experiment Predation of eggs by longnose males has been observed under natural conditions (Bartnik, MS, 1970). In these instances, males with eggs were 107 never seen to eat t h e i r own eggs, but when t h e i r nest s i t e s were disturbed by s h i f t i n g the substrate other males entered the nest s i t e and devoured exposed eggs. Egg losses due to i n t r a s p e c i f i c predation were also evident i n laboratory tests when the parental male was removed immediately a f t e r spawning, but not when the parental male was present under otherwise i d e n t i c a l conditions (Bartnik, pers. observation). Although the above information might lead one to suspect that the r i s k of egg predation would be great when males are grouped c l o s e l y toge-ther, the r e s u l t s of preceding experiments suggest that quite the opposite may be true. Since males of t e r r i t o r y c l u s t e r s appear to remain f a i t h f u l to t h e i r chosen s i t e s (see page 65 ) and i n t e r a c t i n g parental males show prolonged s i t e attachment, at the nest s i t e ( F i g . 18), i t seems possible that i n t r a s p e c i f i c interference and egg predation would be reduced.in t e r r i t o r y c l u s t e r s . The following experiment compares c l o s e l y spaced groupings of t e r r i -t o r i e s with more widely spaced t e r r i t o r i e s to determine whether the t e r r i -tory c l u s t e r s function to reduce i n t r a s p e c i f i c interference and egg predation. i i . Procedure Two d i f f e r e n t spacings of t e r r i t o r i a l males were induced by placing BC structures, i n which the underlying coarse gravel was 8 cm i n depth, i n e i t h e r a c l u s t e r (mean distance between each - 23 cm; F i g . 27A) or spread out i n the experimental channel (mean distance between each = 93-6 cm; F i g . 27B). Remaining bottom areas were covered with f i n e gravel and i n each channel one BF structure was placed downstream of the clustered 108 A . s til < H • l m 1 1 1 [BC] El IE 1 — B F i g . 27. Arrangement of BC structures i n channel (top view) to induce (A) c l o s e l y spaced or clustered male t e r r i t o r i e s and (B) more widely spaced male t e r r i t o r i e s . Arrows denote d i r e c t i o n of current. 109 structures (Fig. 27A) or midway between, the spread structures (Fig. 27B). In each of three r e p l i c a t e s for each type of spacing, four r i p e males were introduced into the channel and allowed to i n t e r a c t f or 48 hr. Observations showed that during t h i s period a stable t e r r i t o r i a l grouping was formed. At the beginning of the t h i r d night period, an ovulated female and three ri p e 'transient' males from holding tanks were released into the channel. Once spawning occurred.at one of the BC structures, 30 min recordings were made at 0-30 min, 1-1/2-2 hr, and 2-1/2-3 hr. Number of substrate contacts, defined as contact of the snout region with the nest s i t e substrate, were scored f o r both transient and t e r r i t o r i a l males. A substrate contact was considered to be an i n d i c a t i o n of egg predation p o t e n t i a l . Number of agonistic acts won against transients by both the parental male and other t e r r i t o r i a l males were also recorded. A f t e r the l a s t recording, a l l dace were removed, k i l l e d , and preserved i n formalin for stomach analyses. Pumps were disengaged and a count was made of a l l v i a b l e eggs remaining i n the nest. i i i . Experimental F i s h A t o t a l of 48 dace were used. The s i x parental males measured 85 to 93 mm i n fork length, while the remaining t e r r i t o r i a l male dace ranged from 81 to 107 mm i n fork length. The 18 transient males meausred 79 to 100 mm i n fork length and the s i x females 85 to 109 mm i n fork length. Sizes of transient males used i n c l o s e l y spaced and widely spaced groupings did not d i f f e r s i g n i f i c a n t l y (p».05; Kolmogorov-Smirnov two sample t e s t ; S i e g e l , 1956). P r i o r to each t e s t , t r a n s i e n t males were kept i n holding tanks where food was constantly a v a i l a b l e . 110 i v . Results and Discussion In clustered groupings of t e r r i t o r i a l males, entries into the nest s i t e by transient males during which egg predation may have occurred ( i . e . , substrate contacts) were few, and almost no egg predation occurred (Fig. 28). In these groupings, t e r r i t o r i a l males remained f a i t h f u l to t h e i r chosen s i t e s and frequently defended them against transient males (Fig. 28). In the more widely spaced groupings, considerable egg predation occurred (Fig. 28). Transient males contacted the nest s i t e substrate numerous times and ate 6.3 eggs per transient male (Fig. 28). In one r e p l i c a t e with widely spaced t e r r i t o r i e s , one t e r r i t o r i a l male abandoned h i s t e r r i t o r y to j o i n transient males i n following the female. This male entered the nest s i t e during the spawning act and ate several eggs (see Replicate I I ; Table XII). Although t h i s p a r t i c u l a r male i n i t i a l l y defended h i s t e r r i t o r y against transient males, his behaviour changed to resemble that of a transient male. However, a l l other t e r r i t o r i a l males tested under these more widely spaced conditions displayed strong s i t e f a i t h f u l -ness . They di d not enter the nest s i t e , but remained on t h e i r t e r r i t o r i e s and defended them against trespassing transient males. Parental males of the clustered groupings were required to defend 2 t h e i r nests l e s s frequently (X = 'A'A.5, p<.001) than parental males of the widely spaced groupings (Fig. 28). Furthermore, a s i g n i f i c a n t l y smaller percentage of these defences against transients by parental males of c l u s -2 tered groupings (X = 20.5, p<. 001) were made i n the presence of the female (Table XII). Intrusions by male dace on courting pairs u s u a l l y disrupted I l l 1 8 16 14 12 _2 o 2 io| « a 8 « E Z 61 CLUSTERED 1 rl WIDELY SPACED I transiento'd' D territorial0^5* S parental (f(f substrate agonistic contacts acts won against transient Jl/1 eggs substrate agonistic eggs eaten contacts acts won eaten against transientJcP F i g . 28. Mean number of substrate contacts, agonistic acts won against transient males, and eggs eaten by d i f f e r e n t types of male dace at nests spawned i n clustered and widely spaced t e r r i -t o r i a l groupings. Replicates I-III are combined. For both experimental groupings, N = 9 for transient and t e r r i t o r i a l males, and N = 3 for parental males. Table XII. Number of substrate contacts and eggs eaten by d i f f e r e n t types of dace at nests spawned i n (A) c l o s e l y spaced, and (B) widely spaced t e r r i t o r i a l groupings. Number of agonistic acts won against transient males and number of v i a b l e eggs remaining i n the nest at the completion of tes t s are also shown for each r e p l i c a t e . Spacing Pattern of T e r r i t o r i e s # Substrate Contacts by trans males t e r r males # Agonistic Acts Won Against trans males by t e r r males par males Closely Spaced (Clustered) # Eggs Eaten by trans t e r r par par tmalesmalemalesrr immales female # Eggs L e f t i n Nest Replicate I 2 0 21(1) 14(2) 2 0 0 0 Replicate II 0 1 36(4) 9(0) 0 0 0 0 Replicate I I I 7 0 23(4) 18(4) 0 0 0 0 Mean No. per male 1/ 0.1/ 8.8(.l;)/ 13.6(2)/ 0.2/ trans male te r r male te r r male par male trans male B. Widely Spaced Replicate I 17 0 18(6) 16(5) 26 0 0 0 Replicate II 12 19* 10(2) 33(19) 12 19* 0 0 Replicate I I I 21 0 11(1) 29(11) 19 0 0 0 Mean No. per male 5.5/ 2.1/ 4.3(1)/ 26(11.6)/ 6.3/ 2.1/ trans male t e r r male t e r r male par male trans male t e r r male 254 289 427 273 380 208 ) = i n presence of female * = a l l by one male M ho 113 the courtship sequence with the female leaving the t e r r i t o r y . This would suggest that interference with spawning a c t i v i t i e s i s reduced i n the c l o s e l y spaced d i s t r i b u t i o n . Observations indicated that transient males could not enter those nest s i t e s surrounded c l o s e l y by other t e r r i t o r i e s as r e a d i l y as they could the more i s o l a t e d ones. I t was quite evident from the observations made that when several male dace intruded simultaneously, the parental male's success at defence was rather poor. Clustered groupings, however, had the e f f e c t of s p l i t t i n g up transient males and e l i m i n a t i n g the "ganging-up" e f f e c t . The behaviour of the t e r r i t o r i a l males i n both types of groupings was, with the one noted exception, s i m i l a r . This suggested that the actual distance between t e r r i t o r i e s was not n e c e s s a r i l y the most c r u c i a l f a c t o r i n determin-ing the success of spawning males. What appeared to be j u s t as important was the type of males occupying a l o c a l area i n the r i f f l e . Proximity of males possibly promotes synchrony through v i s u a l and/or p h y s i c a l contacts. Such synchronized behaviour apparently reduces the l i k e l i h o o d of t e r r i t o r i a l males abandoning t e r r i t o r i e s to i n t e r f e r e with spawning and/or prey on.eggs of neighbouring males. In a d d i t i o n , i t appears that transient males are l e s s l i k e l y to intrude i n t o c l u s t e r s of t e r r i t o r i e s . When receptive females move within a c l u s t e r of t e r r i t o r i a l males, each male remains f a i t h f u l to i t s s i t e . Each t e r r i t o r i a l male defends i t s t e r r i -tory against transient males and intrudes infrequently on neighbouring f i s h . An assemblage of c l o s e l y spaced active males may be of further value i n a t t r a c t i n g receptive females which may spawn with several males i n the c l u s t e r . As each, male spawns, the increased nest s i t e tenacity and egg 114 eating i n h i b i t i o n of parental dace would help to further reduce the l i k e l i h o o d of males i n the c l u s t e r i n t e r f e r i n g with each others spawning a c t i v i t i e s and/or eating each others eggs. Male three-spined sticklebacks well established on a t e r r i t o r y and heavily involved i n parental a c t i v i t i e s also show l i t t l e i n t r u s i o n or egg r a i d i n g behaviour (Wootton, 1971b). Although van den Assem (1967) recognized that females might be more attracted to a group of males than a s o l i t a r y one, he also recognized c e r t a i n disadvantages f o r a stickleback breeding i n a t e r r i t o r y c l u s t e r . Unlike longnose dace, whose t e r r i t o r i e s are f i x e d i n s i z e by the topography of the rock r i f f l e bottom, stickleback t e r r i t o r i e s become reduced i n s i z e when crowded together. As a r e s u l t , stickleback males i n such groupings act as competitors, i n t e r a c t i n g aggressively and i n t e r f e r i n g with one another. Van den Assem found the s t e a l i n g of eggs, i n t e r r u p t i o n of fanning bouts, and i n s u f f i c i e n t development of fanning to be common i n these r i v a l s i t u a t i o n s . The foregoing conclusions on dace t e r r i t o r y c l u sters are strongly supported by a two-part experiment done with s i m i l a r l y c l o s e l y spaced males .eh, The a v a i l a b i l i t y of spawning gravel was used to control the beha-viour of the males. In both experiments A and B, f i v e structures were placed i n the channel (mean distance between each = 30 cm) and the remaining bottom area wasacoveredhwlth-if ±ne'r-gravel'v. eln ;experiment"A?- onevstructure was of the BC type and the other four of the BF type. In experiment B, a l l f i v e structures were of the BC type (Fig. 29A). Thus only one s u i t a b l e area for a male t e r r i t o r y existed i n experiment A, but there were f i v e i n experiment B. Four rip e males and one r i p e female were tested i n each 115 F i g . 29A. Total number of agonistic acts won by male and female. dace i n experiments A and B before and a f t e r spawning. Abbreviations r e f e r to structure-substrate combinations. F i g . 29B. Levels of agonistic a c t i v i t y performed at the nest s i t e i n experiments A and B before and a f t e r spawning. (LD 16:8) 116 experiment. Af t e r a 12 hr adjustment period, four 30 min observations were made during both day and night to record agonistic a c t i v i t y . Once spawning began, two a d d i t i o n a l r i p e males referred to as " t r a n s i e n t s " were added. Two 30 min recordings we-re,mSde,ionedimmediately:'and one .1 hr l a t e r . In experiment A, only one male t e r r i t o r y existed throughout the t e s t , while several existed i n experiment B ( F i g . 29A). Experiment A's one sui t a b l e spawning s i t e was defended over 500 times during the 6 hr of observation. The number of defences ( i . e . , agonistic acts won) at experi-ment B's spawning s i t e , however, was considerably lower ( F i g . 29A). A l -though the l e v e l of agonistic a c t i v i t y was higher i n experiment A (12 to 119/30 min) than i n experiment B (3 to 23/30 min), s i m i l a r trends occurred i n both before and a f t e r spawning (Fig. 29B). The parental male i n experi-ment A was intruded on repeatedly (29 to 72/30 min) as h i s l e v e l of agonistic a c t i v i t y i ndicates ( F ig. 29B) . Eggs were eaten, i n t h i s experiment but not i n experiment B (Table X I I I ) , i n which the parental male received consider-ably fewer i n t r u s i o n s from other males (Fig. 29B). Therefore, differences i n success at nest defence i n the two s i t u a -tions would appear to be a t t r i b u t a b l e to the diffe r e n c e i n the number of intr u s i o n s from other males. Unlike the test males without t e r r i t o r i e s i n experiment A, behaviourally synchronized ones of experiment B spent the majority of time defending t h e i r t e r r i t o r i e s and intruded less upon neigh-bouring males. Experimental males in. experiment A, however, c o n t i n u a l l y entered the BG area before the spawning act and attempted to court and spawn with the female. Consequently, the test males without t e r r i t o r i e s 117 Table XIII. Number of eggs eaten by dace i n Experiments A and B Number of Eggs Eaten Type of Dace Experiment A Experiment B parental male 0 0 parental female 0 0 experimental male 1 0 0 experimental male 2 19 0 experimental male 3 9 0 transient male 1 0 0 transient male 2 0 0 118 i n experiment A were i n r e a l i t y transient males. Although the density of males and the spacing of s h e l t e r s was i d e n t i c a l i n both experiments, a difference i n the success of the spawning males occurred. This difference was r e l a t e d to the behaviour of the males surrounding the spawning s i t e . These r e s u l t s reemphasized the f a c t that the simultaneous i n t r u s i o n of several males was the most d i s r u p t i v e . Under these conditions, the parental male was not able to prevent entry, sub-s t r a t e contacts, and subsequent egg predation. b. S i g n i f i c a n c e of Post Spawning Changes i n Male Behaviour Although evidence already c o l l e c t e d argues that an important function of male t e r r i t o r y i s to provide a s i t e f o r courtship and egg deposition, a possible second function i s investigated. This i n v e s t i g a t i o n was prompted by the f a c t that once t e r r i t o r i a l males have spawned they continue to defend the nest s i t e . It seems l i k e l y that t e r r i t o r i a l i t y by males with eggs, although s t i l l reserving the nest s i t e f or possible future spawnings, would also serve to protect already deposited eggs from i n t r a s p e c i f i c pre-dation. Laboratory observations of numerous males have revealed that both quantitative and q u a l i t a t i v e changes occur i n the behaviour of t e r r i t o r i a l males immediately a f t e r spawning. Levels of a g o n i s t i c a c t i v i t y at the nest s i t e generally peak at t h i s time, and the nature of the aggressive behaviour changes. Observations ind i c a t e that these changes i n male behaviour l a s t from one to several hours. A f t e r that time, parental male behaviour be-comes i n d i s t i n g u i s h a b l e from normal pre-spawning t e r r i t o r i a l behaviour. 119 Changes i n the behaviour of t e r r i t o r i a l f i s h during the reproductive phase are w e l l documented. We know from the work of several authors, e s p e c i a l l y Sevenster (1961) and Wootton (1970), that the behaviour of male three-spined sticklebacks changes s i g n i f i c a n t l y at the time they begin nest-building and again when they f i n i s h n est-building. Further changes i n the behaviour of male sticklebacks are observed a f t e r f e r t i l i z a -t i o n of eggs (Sevenster-Bol, 1962; Black, 1971; Wootton, 1972), with peaks of the U-shaped temporal pattern i n aggressive behaviour c o i n c i d i n g with periods immediately a f t e r egg f e r t i l i z a t i o n and f r y hatching. A d e t a i l e d analysis of the a g o n i s t i c behaviour of a t y p i c a l male dace before and a f t e r spawning i s shown i n Figure 30. An increase i n the frequency of occurrence of the darting behaviour pattern i s observed a f t e r spawning. Before spawning, t e r r i t o r i a l males usually react to an intruding male only once i t enters the t e r r i t o r y . Thus the resident and intruding f i s h are i n close proximity, and the behaviour patterns performed i n defence are usually butting and b i t i n g ( Fig. 30). However, once an attack i s i n i t i a t e d by a t e r r i t o r i a l male i t i s continued u n t i l the intruder i s driven outside the t e r r i t o r y . A f t e r spawning, parental males respond to dace approaching or even s k i r t i n g along the t e r r i t o r y boundary. Parental males dart out towards such intruders. I f the intruder does not f l e e i n response to the owner's dart, but continues to move towards the t e r r i t o r y , then the defending male w i l l b i t e or butt the intruder as i t attempts to cross the border. Once such an attack i s i n i t i a t e d , the parental male often gives chase u n t i l the intruder retreats away from the t e r r i t o r y . With t h i s apparent increase 120 HOURS F i g . 30. Agonistic a c t i v i t y performed by a t e r r i t o r i a l male before and a f t e r spawning. Histograms indi c a t e frequency of . occurrence (%) of d i f f e r e n t a g o n i s t i c behaviour patterns fo r each 15 min recording. Three other males were present i n addition to the spawning p a i r . 121 i n aggressive motivation, trespassing f i s h are attacked more r e a d i l y and l e v e l s of agonistic a c t i v i t y at the nest s i t e are higher r e l a t i v e to l a t e r i n the post spawning period. Stickleback studies (van den Assem, 1967; Black, 1971; Wootton, 1971b, 1972) have shown that t e r r i t o r y s i z e a c t u a l l y increases as a function of aggressive l e v e l s . Sizes of dace t e r r i t o r i e s were not measured i n the present study, since the shelters defended by dace were f i x e d i n s i z e . Unlike stickleback t e r r i t o r i e s , s i z e s of dace t e r r i t o r i e s observed i n nature are f i x e d by the topography of the bottom. Any increases i n dace t e r r i t o r y s i z e which may occur i n the rock r i f f l e habitat would be s l i g h t and d i f f i c u l t i f not impossible to quantify. Topography of the habitat i s known to influence the s i z e or borders of the t e r r i t o r i e s of a number of f i s h species (Greenberg, 1947; F a b r i c i u s , 1951; Kalleberg, 1958). The actual l e v e l of agonistic a c t i v i t y performed by a parental male dace i s , of course, dependent on the number of trespassing or i n t r u d i n g dace i n t e r a c t i n g with the parental male. However, q u a l i t a t i v e changes i n aggressive behaviour, as described here, are common to a l l t e r r i t o r i a l males a f t e r spawning. Furthermore, males observed to spawn several times over a number of days display t h i s behavioural change a f t e r each successive spawning. Therefore, each subsequent clutch of eggs would appear to be afforded the same degree of protection as the f i r s t . A second change i n male behaviour also occurs a f t e r spawning. The rate of substrate probing by parental male dace remains high, but then declines quickly within several hours. Data for t h i s behaviour change were presented e a r l i e r (see F i g . 18). Although substrate probing i s 122 considered to be an important component of courtship behaviour, i t also appears to be important i n nest s i t e preparation and/or maintenance. A more d e t a i l e d analysis of the s i g n i f i c a n c e of t h i s post spawning change i n male substrate probing behaviour follows. c. S i g n i f i c a n c e of Male T e r r i t o r y as Determined by V u l n e r a b i l i t y of Dace Eggs to I n t r a s p e c i f i c Predation i . Outline of Experiment Since post spawning changes i n male dace behaviour are temporary and most pronounced during the f i r s t few hours a f t e r spawning, the l i k e l i h o o d that this period coincides with periods of greatest egg v u l n e r a b i l i t y was examined. The following ser i e s of r e p l i c a t e s tested the v u l n e r a b i l i t y of dace eggs to i n t r a s p e c i f i c predation at varying times a f t e r spawning. i i . Procedure For each r e p l i c a t e , a parental male was provided by introducing a sin g l e male into a small channel (60 x 23 cm). Except for one BC s t r u c -ture, i n which the underlying coarse gravel was 8 cm i n depth, the bottom area was covered with f i n e gravel. At the commencement of a night period, 12 hr a f t e r the male's introduction, an ovulated female was added to the channel. Spawning i n v a r i a b l y took place over the coarse substrate of the BC structure. For each egg v u l n e r a b i l i t y t e s t , three r i p e male dace held i n a transparent p l a s t i c box (12.5 x 7.5 x 7.5 cm) with a p l a s t i c screen top (3 meshes/cm) were placed s l i g h t l y downstream from the spawning s i t e . To commence an egg v u l n e r a b i l i t y t e s t , the screen was removed and these t r a n s i e n t males-were' otales released. V u l n e r a b i l i t y t e s t s were of 3 hr duration 123 with three 30 min recordings made at 0-30 min, 1-1/2-2 hr, and 2-1/2-3 hr. Number of substrate contacts were scored for a l l dace. Substrate probing, of course, was included i n t h i s category. A g o n i s t i c acts per-formed by the parental male were also recorded. After the l a s t recording a l l dace were removed, k i l l e d , and preserved for stomach analyses. Pumps were disengaged and a count was made of a l l v i a b l e eggs remaining i n the nest. V u l n e r a b i l i t y tests were made at four d i f f e r e n t times a f t e r spawning (0,4,24, and 72 hr) and a l l took place during the night period. Times of 0, 4, 24, and 72 hr a f t e r spawning were chosen because of t h e i r p o s i t i o n r e l a t i v e to the period of post spawning changes i n male dace behaviour. The most marked change occurs immediately a f t e r spawning, while 4 hr f a l l s somewhere a f t e r t h i s period. Twenty-four hr. i s w e l l outside t h i s period as i s 72 hr. A shortage of both time and f i s h prompted the d e c i s i o n not to r e p l i c a t e the 72 hr t e s t . For the three r e p l i c a t e s at 0 hr a f t e r spawning, the p l a s t i c box con-t a i n i n g transient dace was placed i n the channel sh o r t l y a f t e r the f i r s t courtship behaviour patterns were performed by the female. Once spawning began, transients were released. Thus the transient f i s h were present during the spawning acts. In the remaining sets of r e p l i c a t e s , the female was removed as soon as her abdomen was noticeably reduced i n s i z e . She was stripped of any remaining ri p e eggs and was placed i n a 40 1 aquarium. This tank always held some ovulated females. The box containing transient dace was placed i n the channel 2 hr before the transient males were to be released. Just before e i t h e r 4, 24, or 72 hr had elapsed since spawning 124 occurred, the parental ( i . e . , r e c e ntly spawned) female was taken from the holding aquarium, checked f o r complete s t r i p p i n g , and returned to the channel. This was followed by the release of the transient males approximately 5 min l a t e r . Parental females were stripped of r i p e eggs to ensure that no further spawning took place a f t e r t h e i r return to the channel. Such females were held i n aquaria with ovulated females since i t was observed e a r l i e r that females and even males kept i n such water were a t t r a c t i v e to t e r r i t o r i a l males ( i . e . , there was an increase i n the courtship l e v e l ) . These f i n d -ings, along with observations of r i p e males synchronizing movements with ovulated females while on the opposite side of an opaque p a r t i t i o n , suggest that an o l f a c t o r y stimulus may induce sexual responsiveness i n male dace. In the g o l d f i s h , the b r i e f i ntroduction of a recently ovulated female g o l d f i s h into water containing males increases the courtship l e v e l of males which p e r s i s t s even i n the absence of the female (pers. comm., N. Stacey). Male channel c a t f i s h demonstrate a strong a t t r a c t i o n to the source of a sexual pheiBmptfereleased bv r i p e females of that species (Timms and Kleerekoper, 1972). The importance of pheromones i n the s o c i a l behaviour of yellow bullheads also has been demonstrated (Todd e_t a l . , 1967). i i i . Experimental F i s h A t o t a l of f o r t y males and ten females were used. Parental males (10) measured 87 to 96 mm i n fork length, while parental females (10) measured 81 to 101 mm i n fork length. Transient males (30) ranged from 75 to 101 mm i n fork length. Before each t e s t , transient males were held i n holding 125 tanks with food constantly a v a i l a b l e . i v . Results and Discussion The procedure used proved to be a s a t i s f a c t o r y method for a t t r a c t i n g transient dace to the nest s i t e where egg predation could occur. When the spent female was returned to the channel i n the 4, 24, and 72 hr t e s t s , she attempted to enter the only a v a i l a b l e enclosure ( i . e . , the nest s i t e ) . The parental male immediately began courting her by frequent substrate probing, trembling, and nudging. The.released transient males were also attracted to the female and courted her by following, nudging, and quiver-ing behaviour patterns. These transient males frequently entered the nest s i t e where the parental male was courting intensely. The parental male frequently interrupted h i s courtship a c t i v i t i e s to defend the nest s i t e against the intruding males. In the presence of the female and the sub-str a t e probing parental male, transient males also probed i n the substrate. Transient males performed considerably more.substrate contacts during ac-t u a l spawning ( i . e . , 0 hr) than they d i d for the other t e s t times ( F i g . 31). This i s a t t r i b u t a b l e , at l e a s t i n part, to the increased stimulus of spawning behaviour, during which both the parental male and female d i s -played considerable substrate probing (Table XIV). During 4, 24, and 72 hr t e s t s , snout contacts with the nest s i t e substrate (used as an i n d i c a t o r of egg predation potential) though les s frequent,were s t i l l performed by transient males. The r e s u l t s i n d i c a t e that eggs were most vulnerable to predation immediately a f t e r being spawned, less so a f t e r 4 hr, and apparently not at a l l i n 24 and 72 hr old nests ( F i g . 31). Since a comparatively large number of eggs were present i n a l l t e s t s (Table XIV) and the l i k e l i h o o d of 126 Hours After Spawning g. 31. Number of eggs eaten and substrate contacts made by transient males at 0, 4, 24, and 72 hr old nests. V e r t i c a l l i n e s above and below means (#,A ) represent ranges. Sample s i z e i s three except for 72 hr where N = 1.. Table XIV. Number of substrate contacts made and eggs eaten by tr a n s i e n t and parental dace at 0, 4, 24, and 72 hr old nests. Amount of ag o n i s t i c a c t i v i t y performed by parental males during test periods and number of v i a b l e eggs remaining i n nests at com-p l e t i o n of v u l n e r a b i l i t y tests are also shown for each r e p l i c a t e . Hours A f t e r # Ago n i s t i c Acts # Substrate Contacts by # Eggs Eaten by # Eggs L e f t eggs spawned by par maler trans males par male £ par females trans males par male par i n Nest female I II III Mean I II III Mean I II III Mean I 32 46 28 24 18 30 31 19 14 14 34 60 18 37.3/replicate 15 20 17 17.3/replicate 20 32 39 32 29 19 11 18 7 12/replicate 12 24 33 20 11 17 19 23 2 0 5 0 3 0 36 0 18 0 14 0 22.7/replicate 14 0 17 0 4 0 11.7/replicate 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 239 362 177 395 270 388 410 187 201 273 128 l o c a t i n g eggs existed, as i n d i c a t e d by substrate contacts, differences i n a c c e s s i b i l i t y appears to have accounted for varying amounts of predation observed. Previous observations have shown that nests attended by parental males f o r longer periods d i f f e r i n the t o t a l amount of substrate probing the gravel containing the eggs has undergone. Differences i n appearance of nests of varying ages are apparent when viewed from above with a face mask. In the f i e l d , older nests generally are m o r e . d i f f i c u l t to locate by means of mask and snorkel viewing than are f r e s h l y spawned ones. In new nests, several eggs adhering to the surface layer of rocks are usually v i s i b l e . However, i n older nests, eggs are located on the under surfaces of stones and down among the i n t e r s t i c e s of gravel. In these older nests, surface pieces of gravel generally must f i r s t be moved before any eggs are. v i s i b l e . Under stream tank conditions, i t has been observed that movements of attending parental males ( i . e . , substrate probing and f i n scraping over the nest substrate) frequently break adhesive points of newly attached eggs, causing them to f a l l deeper into crevices. Since the period of heightened male a c t i v i t y (see Post Spawning Changes i n Male Behaviour, page 118 ) i s most marked within the f i r s t few hours a f t e r spawning, i t apparently serves to bury f r e s h l y deposited eggs deeper i n the substrate. Although egg v u l n e r a b i l i t y tests suggest that eggs are s t i l l vulnerable 4 hr a f t e r spawning, they are considerably l e s s so than immediately afterwards. The p o s s i b i l i t y of changes i n egg odor being correlated with v u l n e r a b i l i t y has not been examined. Although eggs i n older nests (24 and 72 hr) were shown not to be vulnerable i n these t e s t s , such eggs are eaten i f the nest i s 129 p h y s i c a l l y disrupted and the eggs are exposed (observed under both f i e l d and laboratory conditions). The r e s u l t s lead one to conclude that dace eggs are most vulnerable to i n t r a s p e c i f i c predation from 0 to 4 hr a f t e r spawning. V u l n e r a b i l i t y then decreases to a very low l e v e l within 24 hr. The period of heightened parental male aggressiveness, frequent substrate probing, and increased s i t e f a i t h f u l n e s s coincides c l o s e l y with t h i s period of v u l n e r a b i l i t y . Furthermore, as mentioned e a r l i e r , post spawning changes i n male behaviour are renewed with each successive spawning bout. Thus the data suggest that parental male t e r r i t o r i a l behaviour, along with i t s r e l a t e d a c t i v i t i e s , renders the eggs i n the nest le s s vulnerable to i n t r a s p e c i f i c predation. d. Other Predators To determine whether dace are preyed on by the other r i f f l e i n h a b i t i n g species of f i s h i n the Alouette River, a c o l l e c t i o n of s c u l p i n s , Cottus  asper and C_. a l e u t i c u s , (90 -to 155 mm i n fork length) , j u v e n i l e steelhead trout, Salmo g a i r d n e r i , (83 to 110 mm i n fork length), and dace (41 to 94 mm i n fork length) were held together under simulated r i f f l e conditions. I r r e g u l a r l y spaced observations were made during both day and night periods over a 2 wk period. i . Trout Juvenile steelhead trout generally maintained p o s i t i o n off the bottom feeding on d r i f t i n g objects. They were never observed to pick stationary items o f f the substratum. The only i n t e r a c t i o n observed between trout and dace consisted of a parental male dace b i t i n g the caudal f i n of trout which 130 d r i f t e d downstream into the male's t e r r i t o r y . A f t e r each bite'the ; trout moved upstream. i i . Sculpins In the simulated r i f f l e , adult sculpins captured and consumed j u v e n i l e dace and on occasion pursued and attacked adult dace. The larger adult dace (80 to 94 mm i n fork length), although often i n j u r e d by such attacks, were never consumed. More s e r i o u s l y injured f i s h , however, f e l l prey to cray-f i s h i n the tank. For the most pa r t , sculpins showed no apparent a t t r a c t i o n towards t e r r i t o r i a l adult dace or towards a defended nest s i t e . Both laboratory observations and analyses of stomachs of f i e l d c o l l e c t e d s c u l -pins have shown aquatic insect larvae to be t h e i r predominant prey. Upon entering a dace nest s i t e , sculpins were never observed to orient to or feed upon the buried eggs.' Parental male dace occasionally butted or b i t a s c u l p i n which entered the nest s i t e , but such agonistic behaviour was mild, not p e r s i s t e n t , and generally unsuccessful i n removing scu l p i n s . Although t e r r i t o r i a l dace were often observed to share an enclo-sure with a s c u l p i n , conspecific males were always attacked when attempting to enter the same s i t e . On s i x separate occasions, a s c u l p i n and a male dace attempted to enter the t e r r i t o r y of a male dace simultaneously. Each time the parental male attacked the dace, but not the s c u l p i n . Since we know that longnose eggs are eaten by c o n s p e c i f i c s , i t would appear that the main danger to a dace t e r r i t o r y and to any eggs within the t e r r i t o r y comes from other dace rather than other species. Wootton (1970) comes to a s i m i l a r conclusion about sticklebacks. He states that under such circumstances one would expect to f i n d that conspecific males are attacked more r e a d i l y than other f i s h . 131 i i i . Egg Predation I n i t i a l l y i t was thought that sculpins and/or trout might prey on dace eggs, but neither f i e l d data nor laboratory observations have sub-stan t i a t e d t h i s . In the laboratory, sculpins never attempted to burrow i n substrate containing dace eggs nor did they appear to be attracted to such s i t e s . Trout held with dace d i d not feed o f f the bottom but concen-trated on d r i f t . F i e l d data on species which I suspected might be egg predators was c o l l e c t e d by examining the stomachs of f i e l d c o l l e c t e d f i s h f o r the pre-sence of eggs. In the spring of 1971, when f r e s h l y spawned eggs were abundant i n r i f f l e s , s c u lpins, trout, and dace were seined. The c o l l e c t e d f i s h were k i l l e d i n s t a n t l y by immersing them in t o formalin and they were returned to the laboratory f o r stomach analyses. At the same time, a sam-ple of several hundred eggs was taken from the entire c o l l e c t i o n area and was returned to the laboratory f o r incubation. A f t e r hatching, f r y were reared u n t i l a p o s i t i v e i d e n t i f i c a t i o n could be made (20 to 30 mm). The re s u l t s showed that the ent i r e sample was comprised of Rhinichthys eggs. This same procedure ( i . e . , c o l l e c t i o n of f i s h e s and eggs) was repeated i n the e a r l y spring of 1972, a f t e r a spawning run of Peamouth chub, Mylocheilus  caurinum, had occupied a section of r i v e r immediately upstream of a large r i f f l e . Unlike dace, the chub i s a broadcast spawner r e l e a s i n g large masses of eggs in t o shallow areas near shore where they adhere to the gravel and rock bottom. However, the current c a r r i e d much of the chub spawn into the r i f f l e area. Here i t attached to rocks, l i k e mats of algae. Unlike the previous year's sample of eggs, a l l eggs taken from the c o l l e c t i o n r i f f l e i n 1972 and reared i n the laboratory were i d e n t i f i e d as peamouth chub eggs. 132 Differences between the two springs i n the number of s c u l p i n and trout stomachs containing eggs would appear to be a t t r i b u t a b l e to the d i s t r i b u t i o n of eggs i n the r i f f l e substrate. When Rhinichthys eggs were abundant (1971) , none of the sculpins and trout examined were found to contain eggs (Table XV). However, when Mylocheilus eggs were abundant (1972), 71.4% of sculpins and 80% of trout examined contained eggs (Table XV). Individual sculpins contained up to 65 eggs. The four trout with eggs produced one, two, three, and nine eggs which suggests they may have been taken as d r i f t . The r e s u l t s therefore suggest that dace spawning habits appear to be e f f e c t i v e i n rendering eggs ina c c e s s i b l e to s c u l p i n and trout predation. Peamouth eggs, however, which f o r the most part were exposed, were accessible to bottom feeding sculpins and to trout as d r i f t . Hunter (1959), Patten (1962), and P h i l l i p s and C l a i r e (1966) have a l l reported s c u l p i n predation on salmonid f r y . Clary (1972) exposed slimy sculpins to brown trout eggs e i t h e r scattered over the bottom or buried under a r t i f i c i a l redds. He found sculpins never preyed on non-hatching trout eggs, but preyed co n s i s t e n t l y on hatching eggs and sac f r y . Since his analyses of stomachs of f i e l d c o l l e c t e d sculpins v e r i f i e d h i s experi-mental findings, Clary suggested that the probable mechanism permitting sculpins to locate redds i s o l f a c t i o n , triggered by some compounds released by hatching eggs or emerging f r y . T e r r i t o r i a l behaviour of parental dace ceases w e l l before hatching occurs and emerging dace f r y are not afforded any p r o t e c t i o n from predation through parental t e r r i t o r i a l i t y . No f i e l d evidence of dace f r y predation by sculpins has been found. This aspect of predation was not pursued 133 Table XV. Frequency of occurrence of eggs i n Cottus asper, C_. a l e u t i c u s , and Salmo ga i r d n e r i stomachs when only Rhinichthys eggs (1971) or Mylocheilus eggs (1972) were abundant i n r i f f l e s from where sculpins and trout were c o l l e c t e d . Numbers i n parentheses are percentages of the number of f i s h examined. Table XVI. Frequency of occurrence of eggs i n Rhinichthys cataractae stomachs when Rhinichthys eggs were abundant i n r i f f l e s from where dace were c o l l e c t e d (1971). The number i n parentheses i s the percentage of the number of dace ex-amined . Table XV Rhinichthys Eggs Abundant (1971) Mylochellus Eggs Abundant (1972) # stomachs # stomachs fork length cocontaining fork length containing Species # examined range (mm) eggs # examined range (mm) eggs Cottus asper male 8 female 1 Cottus aleuticus male 4 female 2 Total Cottus sp. 15 Salmo gairdneri 3 93-130 159 98-112 110-118 80-119 0 0 0 0 0(0) 0(0) 14 2 5 0 21 5 78-132 82-107 90-111 84-130 12 1 2 0 15(71.4) 4(80) Table XVI # stomachs Dace # examined fork length range (mm) containing eggs Adult male 27 75-95 1 Adult female 24 77-110 0 Juveniles 8 52-71 0 T o t a l 59 1(1.7) 134 further as i t i s somewhat superfluous to the o v e r a l l study. Stomach content data of f i e l d c o l l e c t e d dace i n d i c a t e that i n t r a -specif i c egg predation i n nature i s s l i g h t . Of 59 dace stomachs examined only one contained eggs (Table XVI). However, both laboratory and f i e l d observations reported herein indicate that i n t r a s p e c i f i c egg predation does occur. e. Significance of Female T e r r i t o r y as Determined by Changes i n Blood Lactate i n Dace A f t e r Exercise I f dace experience some form of stress during prolonged periods of swimming i n or holding p o s i t i o n against strong currents, p r o v i s i o n of a s h e l t e r from current would be an advantageous consequence of t e r r i t o r i a l i t y . Acting on this conjecture, an i n i t i a l set of experiments was conducted to determine whether dace without access to areas of reduced flow experienced greater weight los s (assumed to be a v a l i d i n d i c a t o r of energy expenditure) than 1) dace with access to such areas, and 2) co n t r o l dace which were i n s t i l l water. No s i g n i f i c a n t differences i n weight loss were revealed i n tests made over 24 hr, 48 hr, 3 days, and 7 days. However, behavioural differences between dace with access and those without access to areas of reduced flow were apparent. No access dace usually ceased swimming a f t e r the f i r s t or second day, and remained pressed against the sides of the tank. I t was suspected that these behavioural changes under no access conditions may have confounded the measures of energy expenditure. P h y s i o l o g i c a l findings by Erickson (1967) and Stevens (1968, 1972) have suggested that weight los s i s a poor i n d i c a t o r of exercise or stress i n freshwater f i s h e s . Apparently both handling and exercise can cause small but consistent increases i n body weight as a r e s u l t of an increase 135 i n osmotic movement of water into the animal. In the following experiment, the s i g n i f i c a n c e of maintaining a sheltered s i t e i n the r i f f l e environment was tested by examining the e f f e c t s of muscular a c t i v i t y on dace blood physiology. i . Outline of Experiment Several authors (Parker and Black, 1959; Parker et a l . , 1959; Beamish, 1966) have observed a c o r r e l a t i o n between mortality and blood l a c t a t e . They have suggested that many f i s h die from fatigue, the l e t h a l agent being l a c t i c a c i d , i t s action di r e c t e d against the blood ph. Movements i n l o t i c environments require active swimming against the current. Fishes accumulate l a c t i c a c i d i n t h e i r tissues very r a p i d l y so they t i r e e a s i l y ; they thus need shelter f a i r l y often, even though they can swim well i n short bursts. M i l l e r (1958) observed that newly i n t r o -duced trout encountered competition from resident trout and were forced to remain i n the current. Here they suffered from accumulation of blood l a c t a t e due to the muscular e f f o r t i n maintaining t h e i r p o s i t i o n . He suggested that they died of simple exhaustion r e s u l t i n g from continuously being chased from occupied t e r r i t o r i e s . Symons (1971) suggested that salmon parr i n t r a n s i t between t e r r i t o r i e s are probably more susceptible than are t e r r i t o r i a l parr to mortality through a build-up i n l a c t i c acid or through predation. Jenkins (1969) found that stable ( i . e . , t e r r i t o r i a l ) brown and rainbow trout were able to avoid excessive swimming. In an e a r l i e r experiment, i t was demonstrated that female dace not defending a t e r r i t o r y against other f i s h are often forced to make frequent movements within the r i f f l e . Since experimental female dace defended only 136 areas of reduced water v e l o c i t y , the p o s s i b i l i t y that such behaviour functions to prevent s t r e s s f u l conditions was tested. i i . Procedure The test chamber or swim tunnel resembled c l o s e l y i n design that used by Kutty and Saunders (1973) to measure swimming of young salmon. It consisted of a c y l i n d r i c a l p l e x i g l a s tube (7.5 cm diam) with a removable s t a i n l e s s s t e e l screen (1.6 meshes/cm, 0.12 cm diam) over the upstream opening. E l e c t r i f i e d (2-3 v o l t s ) s t a i n l e s s s t e e l bars (0.12 cm diam, spread 0.6 cm apart) covered the downstream opening (Fig. 32). The swim tunnel was submerged i n the stream tank containing water at 14 + 2C. Each test f i s h was introduced into the tube and the screen placed over the upstream opening. A f t e r a short adjustment period (2-3 min), the t e s t f i s h was exercised for 5 min. Water was passed through the tube by two water pumps, each with a capacity of 2530 1/hr. Preliminary work determined 5 min to be the maximum time dace would a c t i v e l y r e s i s t shock by swimming against the current. Actual flow passing through the tube varied between 51 cm/ sec and 87 cm/sec, depending on whether one or two pumps were engaged. Although longnose dace have a remarkable a b i l i t y to remain stationary on the bottom i n flowing water, both the curvature of the tube and the occasional use of increased flow ( i . e . , the second pump engaged) prevented them from so doing. When dace stopped swimming vigorously, they were swept downstream by the current. Upon contacting the e l e c t r i f i e d bars, dace received a shock. When experimental f i s h were noticeably fatigued ( i . e . , d i s o r i e n t e d ) , they avoided swimming and gained r e s t i n g time by remaining pressed against the e l e c t r i f i e d bars. During instances of prolonged 55 cm F i g . 32. Diagram of swim tunnel used to exercise dace. 1 - p l e x i g l a s tube; 2 = i n l e t s from pumps; 3 = screen; 4 = e l e c t r i f i e d bars. Wires from bars to e l e c t r i c a l source have been omitted. 138 contact with the e l e c t r i f i e d g r i d (>5 sec), the e l e c t r i c current was turned o f f and dace were allowed to move o f f the bars. To compare blood l a c t a t e l e v e l s of exercised and non-exercised dace, a c o n t r o l group was included to ascertain a r e s t i n g state. These dace were placed i n the swim tunnel as outlined above, but with the v e l o c i t y of water passing through decreased to 2 cm/sec. These dace were not forced to swim and generally remained on the bottom. Treatments were otherwise i d e n t i c a l . A f t e r the test period, dace were removed from the swim tunnel, anesthetized with MS-222, and blood samples taken. F i s h were sectioned at the caudal peduncle and blood was c o l l e c t e d i n heparinized c a p i l l a r y tubes. Since dace contain only a small volume of blood, i t was necessary to pool samples from two test f i s h f o r blood l a c t a t e analyses. Two parts of 8% p e r c h l o r i c a c i d were added to one part blood, the whole blood was centrifuged (2000 rpm, 10 min), and the serum was held i n r e f r i g e r a t e d b o t t l e s . Analysis of l a c t a t e was made spectrophotometrically. The procedure used was that published by the Boehringer Mannheim Co. (BMC TLAA 2972). One a l t e r a t i o n was made. In order to obtain r e p l i c a t e s of samples, serum samples were di l u t e d i n a 1:1 r a t i o with d i s t i l l e d water before analysis. i i i . Experimental Fish Twenty-four dace (both sexes represented) measuring 77 to 95 mm i n fork length were used. Since demands f o r breeding dace were great, only dace i n post spawning condition were s a c r i f i c e d . A l l dace had been starved for 24 hr before t e s t i n g . 139 i v . Results and Discussion Differences between l a c t a t e values of control (non-exercised) and experimental (excercised) f i s h (Table XVII) were compared with the 1 - t a i l e d Mann-Whitney U test ( S i e g e l , 1956) and found to be s i g n i f i c a n t l y d i f f e r e n t (p = .008). Since a l l dace were handled i d e n t i c a l l y with exception of the excercise period, observed differences could not have been due to handling but must r e f l e c t a r e a l difference i n excercise s t r e s s . Several dace re-turned to holding tanks suffered no l a s t i n g i l l - e f f e c t s . Therefore, l a c t a t e build-ups reached by exercised dace were obviously less than l e t h a l l e v e l s . In salmonid f i s h e s , r e s t i n g l a c t a t e l e v e l s vary from approximately 5 to 24 mg% (Parker et a l . , 1959; Black, 1955, 1956, 1957 abc). A f t e r 15 min of vigorous exercise, Black (1955) found blood l a c t a t e i n Kamloops trout yearlings varied from 99.5 to 100.2 mg%. Lactate l e v e l s of 125 mg% for chinook salmon (Parker and Black, 1959) and 100 mg% f o r haddock (Beamish, 1966) have been suggested as being l e t h a l . The small amount of blood i n dace necessitated a rather crude extrac-ti o n method. Therefore, what i s l i s t e d i n Table XVII as blood l a c t a t e i s i n r e a l i t y some combination of a r t e r i a l and venous blood plus a small amount of body f l u i d s . These sources of error are one possible reason for the seemingly high control l e v e l of l a c t a t e (Table XVII). A second explanation may be that permanently high l a c t a t e l e v e l s are common to dace. Hochachka (1961) showed that rainbow trout conditioned to fast water showed such high l e v e l s of blood l a c t a t e . In any case, neither exercised nor control means should be regarded as absolute terms. What, however, can be accepted possibly from the r e s u l t s i s that l a c t a t e appears to be higher i n exercised dace, whether i n terms of blood or muscle l a c t a t e . 140 Table XVII. Blood l a c t a t e values for (A) Non-exercised and (B) Exercised dace. Blood l a c t a t e i s expressed i n milligrams l a c t a t e per 100 ml whole blood (mg % ) . Fork Time Time Total Time Blood Wt. Length Swimming Resting i n Tunnel Lactate Dace (gm) (mm) (sec) (sec) (sec) (mg%) A. NON-EXERCISED (CONTROL) 1 2.6 79 _ 300 48.3 2 6.5 90 - - 300 3 4.1 78 _ 300 48.4 4 7.0 95 - - 300 5 6.2 86 _ 300 36.8 6 4.7 86 - - 300 7 5.3 85 _ _ 300 35.3 8 4.5 80 - - 300 9 4.7 85 _ 300 47.3 10 5.7 87 - - 300 11 6.6 88 _ _ 300 41.3 12 5.3 85 - - 300 x= 5.3 x- 85.3 x= 42.9 (gm) (mm) (mg%) B. EXERCISED 1 5.3 81 275 25 300 46.3 2 6.1 85 259 41 300 3 5.8 84 199 101 300 70.3 4 4.1 75 238 62 300 5 5.8 87 250 50 300 71.3 6 4.1 77 187 113 300 7 4.1 79 151 149 300 73.8 8 4.9 81 242 58 300 9 5.0 89 147 153 300 60.7 10 6.2 90 253 47 300 11 5.2 80 138 162 300 60.7 12 8.5 95 182 118 300 x= 5.5(gm) x= 83.6 (mm) . x= 210(sec) x= 89.9(sec) x= 63.8(mg%) U - 3, p - .008 Blood l a c t a t e l e v e l s of exercised dace are s i g n i f i c a n t l y greater than those of non-exercised dace. ( 1 - t a i l e d Mann Whitney U t e s t ) . Kp-1, p » .05 Body weights of exercised and non-exercised groups of dace did not d i f f e r s i g n i f i c a n t l y . (Kolmogorov-Smirnov two sample t e s t ) . 141 Observations of i n d i v i d u a l s being exercised i n the swim tunnel indicated that dace displayed an i n a b i l i t y to a c t i v e l y swim into the current for the duration of the 5 min test period. Exercised dace showed v i s i b l e signs of fatigue as they l o s t t h e i r a b i l i t y f o r co-ordinated locomotion. They often turned v e n t r a l side up and were swept sideways against the downstream bars. Upon contacting the bars, dace usually re-mained i n a semi-circular posture, making rapid r e s p i r a t o r y movements. A s i m i l a r state of fatigue i n nature may render dace vulnerable to predation. Such dace may be swept downstream into open areas or pools. Dace unable to make rapid co-ordinated movements may be susceptible to s c u l p i n and even c r a y f i s h predation. An example of the l a t t e r , i n v o l v i n g an injured adult dace, has been observed i n the stream tank. Healthy dace able to react quickly to s t a l k i n g c r a y f i s h were never captured. The r e s u l t s show that dace cannot hold against strong currents (51-87 cm/sec) f o r any great length of time. Dace forced to do so experience a r i s e i n blood l a c t a t e , and must sooner or l a t e r pay o f f t h i s oxygen debt. The f a c t that dace 'give up' and f a l l back may or may not be associated with the blood l a c t a t e l e v e l s recorded. Dace which become fatigued, d i s o r i e n t e d , and drop downstream may be vulnerable to predation. Thus a possible function of t e r r i t o r y i n both male and female dace i s that i t provides a refuge from the current. f. T e r r i t o r i a l Behaviour as a Dispersing Mechanism i . Outline of Experiment Although breeding dace aggregate i n c l u s t e r s ( F i g . 17), they continue to defend i n d i v i d u a l t e r r i t o r i e s . Thus i t seems possible that the number 142 of dace breeding within l o c a l i z e d areas might be l i m i t e d i f s u i t a b l e space i s i n short supply. The following experiment was conducted to determine whether t e r r i -t o r i a l behaviour acts as a mechanism for dispersing surplus f i s h away from densely populated areas. Breeding ( t e r r i t o r i a l ) and non-breeding (non-t e r r i t o r i a l ) dace were tested i n an experimental channel i n which the density of dace was increased i n a sequential fashion. The r e p l i c a t e with non-breeding dace was included for comparative purposes. i i . Procedure The experimental set up consisted of a " r i f f l e " zone and a deeper slower flowing "pool" zone ( F i g . 33A). A brown rubber mat was used to create the sloping f l o o r between the r i f f l e and pool. The r i f f l e area held s i x uniformly spaced enclosures (BC) with coarse gravel covering the remaining bottom area. The pool area was devoid of any overhead cover or s h e l t e r components, containing only a layer of f i n e gravel. Both f i e l d and laboratory observations have shown that dace generally avoid this type of habitat. Both r e p l i c a t e s were 4 days i n duration. Six dace (three male, three female) were introduced into the channel i n i t i a l l y and an a d d i t i o n a l s i x (three male, three female) were added on each of the second, third,and fourth days. Recordings were begun 12 hr a f t e r each group was introduced. During the four day and four night 30 min observations, records were kept of 1) p o s i t i o n s of i n d i v i d u a l dace at 15 min i n t e r v a l s , and 2) number and l o c a t i o n of a g o n i s t i c acts won by s o l i t a r y and grouped dace. Dace were scored as occupying the pool zone i f they came i n contact with the pool sub-str a t e or swam about i n the water column of the pool zone for a minimum of 10 sec. 143 F i g . 33 A. Diagram of experimental channel i l l u s t r a t i n g r i f f l e and pool zones. F i g . 33 B. Group sizes and d i s t r i b u t i o n of dace at increasing d e n s i t i e s during breeding and non-breeding phases. Above: Percent occurrence of dace i n enclosures (BC), open areas (0), and pool (P). Below: Percent occurrence of d i f f e r e n t group s i z e s . Percent occurrence i s c a l c u l a -ted as shown on pages 46 and 50. B. 90 60 30 « 0 UJ oc 3 + O 75 50 25r (Day i) 6 cjace BC O P ii n n (Day 2) 12 dace 11 i i t, BC O P il n J I n (Day 3) 18 dace BC O P (Day 4) 24 dace BC O P (Breeding JNon-Breeding ULal l l l l l . 1 2 3 4 4+ 1 2 3 4 4+ 1 2 3 4 4+ 1 2 3 4 4+ GROUP SIZE 144 i i i . Experimental F i s h The 24 breeding dace (twelve male, twelve female) measured 78 to 106 mm i n fork length. The 24 non-breeding dace (twelve male., twelve female) were c o l l e c t e d i n September and measured 76 to 105 mm i n fork length. i v . Results and Discussion The occurrence of d i f f e r e n t group si z e s of breeding and non-breeding dace on day 1 was consistent with previous findings (compare F i g . 33B with F i g . 24). Both breeding and non-breeding dace were predominant at enclosures (BC) , occurring infrequently i n the pool zone ( F i g . 33B). With the doubling of density, however, breeding dace responded d i f f e r e n t l y from dace i n the non-breeding r e p l i c a t e . S o l i t a r y breeding dace remained the predominant group s i z e with occasional pairs and even fewer groups of three and four. The occurrence of f i s h at enclosures (BC) dropped while the occurrence of f i s h i n the pool rose markedly from 4.8 to 23.3% ( F i g . 33B). In the non-breeding r e p l i c a t e , the occurrence of s o l i t a r y dace f e l l s t e a d i l y , and l a r g e r groupings ( i . e . , three, four,and four plus) predomina-ted as de n s i t i e s were increased. As a r e s u l t , the occurrence of dace at enclosures remained high throughout the 4 day period and no marked increase i n the occurrence of dace i n the pool was observed. In the breeding r e p l i c a t e , only on days 3 and 4 when de n s i t i e s were three and four times the i n i t i a l density d i d a noticeable change i n the group sizes of dace take place. On these f i n a l two days, the occurrence of s i n g l e dace was reduced and l a r g e r group sizes (two, three, four, and four plus) became more frequent. Enclosures were s t i l l occupied, but 145 increasing numbers of dace were found i n open areas between enclosures on days 3 and 4. The occurrence of dace i n the pool zone d i d not increase, but f e l l s l i g h t l y on these l a s t two days. When l e v e l s of a g o n i s t i c a c t i v i t y i n the r i f f l e zone are r e l a t e d to the percent occurrence of dace i n the pool, spacing patterns and d i s t r i b u -t i o n of experimental f i s h are more e a s i l y explained. Breeding i n d i v i d u a l s displayed a marked increase i n l e v e l s of a g o n i s t i c a c t i v i t y with the doub-l i n g of f i s h density (Fig. 34). This increase corresponded with the increase i n occurrence of dace i n the pool zone. With the subsequent density increase, a g o n i s t i c a c t i v i t y rose further. However, there was no increase i n the percent occurrence of dace i n the pool. The f i n a l density change brought about a marked f a l l i n agonistic a c t i v i t y and a decrease i n the percent occurrence of dace i n the pool. Keenleyside and Yamamoto (1962) found aggressiveness of wild salmon to increase and peak at intermediate den s i t i e s but then to decrease at s t i l l higher d e n s i t i e s . Fenderson and Carpenter (1971) reported a s i m i l a r response f o r hatchery A t l a n t i c salmon. Agonistic or t e r r i t o r i a l behaviour of breeding dace appeared to be most e f f e c t i v e i n dispersing surplus i n d i v i d u a l s into the pool zone when the density was doubled. However, t r i p l i n g density reduced the e f f e c t i v e -ness of t e r r i t o r i a l defence and a clumped d i s t r i b u t i o n r e s u l t e d . I t has long been known that s o c i a l structure of a group of animals can be al t e r e d by changing the density of the group. Kalleberg (1958) observed that very high population densities of hatchery trout resulted i n the.disruption of t e r r i t o r i a l behaviour and the formation of schools. Similar changes occur i n n a tural populations of the Japanese salmonid, Plecoglossus, (Kawanabe, 1958). F i e l d data suggest that the d e n s i t i e s of dace produced on day 1 most 146 Relationship between percent occurrence of dace i n pool and mean number of a g o n i s t i c acts won per 30 min at increasing densities during breeding (•) and non-breeding (A) phases. 147 c l o s e l y approximated those i n nature. Those of day 2 are encountered only occasionally. Densities on the t h i r d and fourth days, however, were some-what u n r e a l i s t i c . Outside the breeding season, dace displayed l i t t l e a g o nistic behaviour and the sequential addition of dace to the channel produced no noticeable d i s p e r s a l e f f e c t . However, since the spacing requirements of dace change markedly when they are breeding, the d i s p e r s a l of dace throughout the o p t i -mum habitat a v a i l a b l e may be an advantageous consequence of breeding t e r r i t o r i a l i t y . Chapman (1962), Mason and Chapman (1965), and Hartman (1965) have stated that f or coho salmon aggressive behaviour appears to be a key-f a c t o r i n causing downstream " d r i f t " . A recent study by Sale (1972) has provided evidence which suggests that the agonistic behaviour occurring among members of the pomacentrid f i s h , Dascyllus aruanus, i s responsible for the e f f i c i e n t dispersion of t h i s species over the a v a i l a b l e c o r a l . Observations made immediately a f t e r a d d i t i o n a l groups of dace were added provided some further information. In the breeding r e p l i c a t e , newly introduced i n d i v i d u a l s encountered aggression from resident dace. New dace moved about from enclosure to enclosure u n t i l f i n d i n g a large group of non-aggressive dace which they joined. Keenleyside and Yamamoto (1962) found that less dominant A t l a n t i c salmon schooled i n mid-water and acted as a nucleus to a t t r a c t more of the le s s successful competitors. Thus, the clumping e f f e c t observed here with dace i s somewhat analagous to the s h i f t toward mid-water schooling shown by crowded salmon (Keenleyside and Yamamoto, 1962) and c o r a l reef f i s h (Sale, 1972). On seven separate occasions during day 2 of the breeding r e p l i c a t e , 148 a g o n i s t i c i n t e r a c t i o n s resulted i n the l o s e r swimming downstream to the pool zone. Such d i s p e r s a l of dace into the pool zone of the experimental channel was interpreted as d i s p e r s a l away from already saturated areas. In nature, such d i s p e r s a l could, of course, be e i t h e r upstream, downstream, or l a t e r a l within the r i f f l e or to another r i f f l e area. In the non-breed-ing r e p l i c a t e , newly introduced dace moved from one enclosure to another, encountering l i t t l e or no., aggression from resident f i s h . D. Discussion of the S i g n i f i c a n c e of S o c i a l Organization i n Dace I t has long been stressed that the s o c i a l organization of a species i s fashioned by a complex web of s e l e c t i o n pressures and not by j u s t one or a few (Crook, 1965). Thus the s o c i a l l i f e of the longnose dace as we observe i t , i s a complex response to numerous variables i n i t s environment. Each d i f f e r e n t behavioural response of t h i s species contributes i n some way to i t s o v e r a l l s u r v i v a l . Nice (1941) , Hinde (1956) , Tinbergen (1957), Carpenter (1958), Wynne-Edwards (1962), and Ardrey (1966) among others, have discussed at length the importance of t e r r i t o r i a l i t y . However, the only d e t a i l e d review of t h i s subject i n fishes has been by van den Assem (1967). He i s also one of the few authors who has c a r r i e d out an experimental i n v e s t i g a t i o n of the function of f i s h t e r r i t o r i a l i t y . His laboratory experiments with the three-spined stickleback l e d him to conclude that the s i g n i f i c a n c e of the male stickleback t e r r i t o r y has to be found i n a reduction of interference by conspecifics during reproductive behaviour. Since t e r r i t o r i a l i t y i n longnose dace i s r e s t r i c t e d to the breeding phase, i t would seem f a i r to assume that i t serves a p r i m a r i l y reproductive 149 function. For males, the major functions of t e r r i t o r y appear to be, 1) p r o v i s i o n of space i n which males can court and spawn with females with minimal interference from other males 2) protection of eggs from i n t r a -s p e c i f i c predation, and 3) refuge from current. For females, the data suggest that the functions of t e r r i t o r y are, 1) refuge from current, and 2) refuge from male attacks. Evidence shows that dace without access to a refuge from the current experience s t r e s s f u l conditions which may render them vulnerable to preda-t i o n . In a d d i t i o n , female dace defending a t e r r i t o r y are able to avoid c o n f l i c t s with breeding males ( i . e . , courtship harassment and attacks from males). Male t e r r i t o r i a l i t y along with i t s re l a t e d behavioural a c t i v i t i e s serves to protect f r e s h l y deposited eggs while making them l e s s accessible to predators. Conclusions reached here as to the functions of dace t e r r i t o r i a l i t y would appear to be open to the same c r i t i c i s m that Tinbergen (1968) voiced i n h i s review of van den Assem's stickleback study. Tinbergen's main ob-j e c t i o n was that van den Assem merely demonstrated a set of adaptive cor-r e l a t i o n s within the species rather than a functional r e l a t i o n between t e r r i t o r i a l behaviour and e x t r a - s p e c i f i c pressures. However, I take issue with t h i s view. Surely f a i l u r e to demonstrate e x t r a - s p e c i f i c s e l e c t i o n pressures f o r the evolution of t e r r i t o r i a l behaviour does not n e c e s s a r i l y i n d i c a t e f a i l u r e to demonstrate a function of t e r r i t o r y . I n t r a s p e c i f i c nest i n t r u s i o n behaviour, during which conspecifics enter a nest or nest s i t e where eggs are e i t h e r i n the process of being deposited or are already present, has been described for sticklebacks 150 (Morris, 1952; van den Assem, 1967; Wootton, 1971a), darters (Reeves, 1907), A t l a n t i c salmon parr (Jones and King, 1952), cyprinodonts (Itzkowitz, 1970), and sunfishes (Keenleyside, 1972). Such behaviour i n longnose dace i s d i s r u p t i v e as some intruders ( i . e . , transient or n o n - t e r r i t o r i a l males) i n t e r f e r e with spawning a c t i v i t i e s and eat recently spawned eggs. In longear sunfish, although nest i n t r u s i o n i s performed by males without nesting t e r r i t o r i e s , i t i s also common i n those males with both a t e r r i t o r y and recently spawned eggs (Keenleyside, 1972). Therefore, i t would seem that the breeding system of t e r r i t o r y c l u s t e r i n g i n sunfish and i n longnose dace are associated with very d i f f e r e n t functions. Keenleyside suggests that longear sunfish nest close together so that males can f e r t i -l i z e eggs i n neighbouring nests, thereby increasing the numbers of t h e i r own progeny. By being c l o s e l y spaced, sunfish males can intrude i n t o neighbouring nests without hazard to t h e i r own eggs. The e n t i r e i n t r u s i o n act l a s t s no more than 2-3 seconds. Unlike sunfish, c l o s e l y spaced t e r r i t o r i a l male longnose dace remain f a i t h f u l to t h e i r t e r r i t o r i e s . Interference and egg predation between neighbours i s reduced. Since the degree of i n t e r r u p t i o n during courtship and spawning may be an important factor i n determining where a female chooses to spawn, the reduction i n i n t r a s p e c i f i c interference may be of equal ( i f not greater) importance to egg predationt. i n the evolution of dace t e r r i t o r y c l u s t e r i n g . Clustered t e r r i t o r i a l male dace i n t e r a c t frequently with other males, e s p e c i a l l y during the formation of the c l u s t e r . Such i n t e r a c t i o n with other males arouses t e r r i t o r i a l males sexually ( i . e . , nest s i t e tenacity and 151 substrate probing are interpreted as sexual behaviour). T e r r i t o r i a l s tickleback males intruded on by other male sticklebacks are also sexually aroused (Wilz, 1972). Wilz suggests that male sticklebacks bear some resemblance to females and may s l i g h t l y activate the male stickleback's sexual-control mechanism d i r e c t l y . A s i m i l a r explanation for sexual arousal i n t e r r i t o r i a l male dace seems possible. Sustained sexual arousal ( i . e . , s i t e attachment and substrate probing) i n clustered male dace would appear advantageous i n 1) a t t r a c t i n g receptive female dace, and 2) burying vulnerable eggs within a shorter period of time. The continued presence of an aggressive male on the nest s i t e , of course, i s always a good deferent to trespassing conspecifics l i k e l y to prey on eggs. In some of van den Assem's (1967) experiments, he observed a beha-v i o u r a l difference between s o c i a l and s o l i t a r y stickleback males; the former performed more sexual behaviour. Aubin (1972) found that male r u f f e d grouse clustered i n groups drummed more frequently than did more randomly or uniformly d i s t r i b u t e d males. Since hens are a t t r a c t e d to drumming logs for mating, the more a c t i v e l y drumming clustered males may a t t r a c t more hens. Darling (1952) has long held that a group of t e r r i t o r i e s has more chance of a t t r a c t i n g females than has an i s o l a t e d t e r r i t o r y . Experiments conducted i n the present study have i n d i c a t e d that t e r r i t o r i a l behaviour may l i m i t the number of dace breeding i n l o c a l i z e d areas, and therefore serve as a dispersing mechanism. However, attempts to determine whether t e r r i t o r i a l behaviour of dace l i m i t s breeding stocks and u l t i m a t e l y regulates population s i z e were not undertaken i n the present study. Watson and Moss (1970) have stated that to demonstrate that t e r r i -t o r i a l behaviour l i m i t s a breeding population, i t i s necessary to show that 152 a s u b s t a n t i a l part of the population does not breed. Non-breeders should be capable of breeding i f the more dominant ( i . e . , t e r r i t o r i a l ) animals are removed. In b i r d s , reproductive t e r r i t o r i e s are quite stable over the en t i r e breeding phase and more recent evidence supports the hypothesis of regula-t i o n of numbers by t e r r i t o r i a l behaviour (Watson, 1967; Krebs, 1971). Klomp (1972) provides a comprehensive review of t h i s c o n t r o v e r s i a l subject pertaining to b i r d populations. Unlike b i r d s , the t e r r i t o r i a l phase i n many f i s h species i s b r i e f and successive waves of spawners u t i l i z e the same substrate. Longnose dace f i t i n t o such a group and determination of whether t e r r i t o r i a l behaviour prevents part of the population from breeding would be most d i f f i c u l t . Most of the a v a i l a b l e l i t e r a t u r e on f i s h populations and t e r r i t o r i a l behaviour deals with the regulation of population d e n s i t i e s rather than population s i z e . However, even t h i s evidence of whether or not t e r r i t o r i a l behaviour functions to regulate f i s h d e n s i t i e s i s at odds. Macan (1963) believes that f i s h populations are prevented from becoming too dense by t e r r i t o r i a l i t y . Le Gren (1965), i n f a c t , states that t e r r i t o r i a l behaviour acts as a density-determining mechanism i n salmonids. Trout introduced into an unfamiliar section of stream inhabitated by other trout are known to be displaced downstream by the t e r r i t o r i a l residents ( M i l l e r , 1958; Jenkins, 1969). Van den Assem's (1967) experimental r e s u l t s with three-spined sticklebacks also supports the hypothesis that t e r r i t o r i a l behaviour i s a p o t e n t i a l agent for l i m i t i n g the numbers of breeding i n d i v i d u a l s . On the other hand, other f i s h behaviourists describe the aggressive behaviour 153 mechanism of the medaka (Magnuson, 1962) and the ayu (Kawanabe, 1958) as being too f l e x i b l e to l i m i t density at higher population d e n s i t i e s . Longnose dace have adopted a breeding s o c i a l organization which allows them to breed within densely populated r i f f l e s without deleterious e f f e c t s to reproductive success. Male dace coming into breeding condition at the same time c l u s t e r together. Males of such groupings remain behaviourally synchronized and exclude n o n - t e r r i t o r i a l males. Such n o n - t e r r i t o r i a l or transient males apparently pose the greatest s i n g l e threat to reproductive success through interference and egg predation. Clustered t e r r i t o r i a l males show strong s i t e attachment and i n t e r f e r e l i t t l e with the reproductive ac-t i v i t i e s of neighbouring f i s h . This c l u s t e r i n g phenomenon makes excellent functional sense within the usually densely occupied r i f f l e s . Thus, i t seems most probable that during the evolutionary h i s t o r y of the longnose dace i n t r a s p e c i f i c interference and egg predation have provided a major s e l e c t i o n pressure favouring both male t e r r i t o r i a l i t y and male t e r r i t o r y c l u s t e r i n g . Since s e l e c t i o n favours c h a r a c t e r i s t i c s that maximize an i n d i v i d u a l s contribution to the gene pool of succeeding generations (Williams, 1966), evolution of t e r r i t o r y c l u s t e r i n g can be adequately explained by natural s e l e c t i o n at the i n d i v i d u a l l e v e l . Williams states that one need not look for group functions to explain the functional aspects of s o c i a l nesting. Clustering behaviour i s c l e a r l y adaptive from the standpoint of i n d i v i d u a l genetic s u r v i v a l . Williams suggests that the behaviour of the group i s e a s i l y understood as the s t a t i s t i c a l summation of i n d i v i d u a l adaptation. 154 LITERATURE CITED Abel, E.F. 1961. Freiwasserstudien liber das Fortpflanzungsverhalten des Monchfisches Chromis chromis Linne, einem V e r t r e t e r der Pomacentriden im Mittelmeer. Z. Tierpsychol. 18: 441-449. Anderson, P.K. 1961. 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