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UBC Theses and Dissertations

An examination of the adaptive significance of interpopulation variation in the behaviour of the guppy… Luyten, Peter Henri 1982

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AN EXAMINATION OF THE ADAPTIVE SIGNIFICANCE OF INTERPOPULATION VARIATION IN THE BEHAVIOUR OF THE GUPPY P o e c i l i a r e t i c u l a t a by PETER HENRI LUYTEN 5.Sc. f The U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1977 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department o f Z o o l o g y ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA O c t o b e r , 1982 (c) P e t e r H e n r i L u y t e n , 1982 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of ZOOLOGY The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date • OCTOBER 15, 1982 DE-6 (3/81) i i A B S T R A C T P o p u l a t i o n s o f g u p p i e s ( P o e c i l i a r e t i c u l a t a ) a r e f o u n d i n r i v e r s i n T r i n i d a d w h i c h d i f f e r i n b i o t i c a n d a b i o t i c c h a r a c t e r i s t i c s . G u p p i e s f r o m t h e s e r i v e r s d i f f e r i n a n u m b e r o f b e h a v i o u r a l a n d m o r p h o l o g i c a l c h a r a c t e r i s t i c s . G u p p i e s i n a t u r b i d l o w l a n d r i v e r w e r e f o u n d t o b e m o r e c o h e s i v e a n d i n h a b i t a r e a s c l o s e r t o s h o r e t h a n f i s h i n c l e a r h e a d s t r e a m r i v e r s . M a l e s i n t h e t u r b i d l o w l a n d r i v e r e x h i b i t e d a g r e a t e r f r e q u e n c y o f t h r u s t s i n t h e i r c o u r t s h i p t h a n d i d m a l e s i n t h e h e a d s t r e a m r i v e r s . C o n v e r s e l y , h e a d s t r e a m m a l e s e x h i b i t e d a g r e a t e r f r e q u e n c y a n d d u r a t i o n o f s i g m o i d d i s p l a y s i n t h e i r c o u r t s h i p t h a n d i d l o w l a n d m a l e s . T h e s e d i f f e r e n c e s i n c o u r t s h i p b e h a v i o u r b e t w e e n m a l e s i n h e a d s t r e a m a n d l o w l a n d r i v e r s p e r s i s t e d i n m a l e s b r e d i n t h e l a b o r a t o r y u n d e r i d e n t i c a l c o n d i t i o n s . T h i s s t r o n g l y s u g g e s t s t h a t t h e d i f f e r e n c e s i n b e h a v i o u r a r e a t l e a s t i n p a r t g e n e t i c a l l y d e t e r m i n e d , a n d t h a t t h e r e f o r e t h e b e h a v i o u r s a r e a p r o d u c t o f n a t u r a l s e l e c t i o n . F i v e e x p e r i m e n t s w e r e d e s i g n e d i n o r d e r t o t e s t t h e h y p o t h e s i s t h a t t h e i d e n t i f i e d d i f f e r e n c e s i n c o u r t s h i p b e h a v i o u r e x h i b i t e d b y t h e p o p u l a t i o n s a r e a d a p t a t i o n s , i n p a r t , t o d i f f e r e n c e s i n t h e t u r b i d i t y o f t h e r i v e r s i n w h i c h t h e p o p u l a t i o n s n a t u r a l l y o c c u r . I f t h e y a r e a d a p t a t i o n s , t h e y w i l l c o n t r i b u t e t o t h e m a t i n g s u c c e s s o f m a l e s i n t h e i r p a r t i c u l a r e n v i r o n m e n t s . M a l e s f r o m t u r b i d a n d c l e a r r i v e r s w e r e p l a c e d i n c o m p e t i t i o n f o r m a t i n g w i t h f e m a l e s u n d e r b o t h c l e a r a n d t u r b i d w a t e r c o n d i t i o n s . M a t i n g s u c c e s s w a s i i i d e t e r m i n e d o n t h e b a s i s o f n u m b e r o f i n s e m i n a t i o n s a n d s p e r m c o n t r i b u t e d t o f e m a l e s b y m a l e s o f t h e d i f f e r e n t p o p u l a t i o n s . S p e r m w h e r e i d e n t i f i e d u s i n g a r a d i o a c t i v e l a b e l l i n g t e c h n i q u e a n d s t a n d a r d a u t o r a d i o g r a p h i c m e t h o d s . I n c l e a r w a t e r , m a l e g u p p i e s f r o m a c l e a r h e a d s t r e a m r i v e r w e r e m o r e s u c c e s s f u l a t m a t i n g w i t h t h e i r own f e m a l e s a n d a s s u c c e s s f u l a t m a t i n g w i t h l o w l a n d f e m a l e s a s m a l e s f r o m a t u r b i d l o w l a n d r i v e r . I n t u r b i d w a t e r , m a l e s f r o m t h e t u r b i d l o w l a n d r i v e r w e r e m o r e s u c c e s s f u l a t m a t i n g w i t h t h e i r o w n f e m a l e s a n d e q u a l l y s u c c e s s f u l a t m a t i n g w i t h h e a d s t r e a m f e m a l e s a s m a l e s f r o m t h e c l e a r h e a d s t r e a m r i v e r . T h e s e r e s u l t s a r e c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t a h i g h f r e q u e n c y o f d i s p l a y a n d d i s p l a y s o f l o n g d u r a t i o n i n m a l e c o u r t s h i p a r e a d a p t a t i o n s t o c l e a r w a t e r c o n d i t i o n s . T h e r e s u l t s a r e a l s o c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t h i g h f r e q u e n c i e s o f t h r u s t s i n m a l e c o u r t s h i p i s a n a d a p t a t i o n t o t u r b i d w a t e r c o n d i t i o n s . M a l e s e x h i b i t i n g s i m i l a r c o u r t s h i p b e h a v i o u r s f r o m t w o g e o g r a p h i c a l l y i s o l a t e d h e a d s t r e a m r i v e r s w e r e s i g n i f i c a n t l y m o r e s u c c e s s f u l a t m a t i n g w i t h f e m a l e s f r o m t h e i r own p o p u l a t i o n s . T h i s s u g g e s t s t h a t o t h e r f a c t o r s , s u c h a s c o l o u r o r p h e r o m o n e s , may a l s o b e i m p o r t a n t i n d e t e r m i n i n g m a t i n g s u c c e s s . iv Table of Contents PAGE Abstract i i L i s t of Tables v i L i s t of Figures v i i Acknowledgements v i i i CHAPTER ONE Introduction 1 CHAPTER TWO Review of the Behaviour and Natural Environments of the Guppy I. Behaviour 13 II. The Study Area 19 Physical Parameters of the Study Rivers A. Methods 24 B. Results 26 CHAPTER THREE Geographic Variation i n the Courtship Behaviour of P o e c i l i a r e t i c u l a t a I. Introduction 29 II. Behaviour i n the Natural Environments Methods 31 A. General behaviour 33 B. Male courtship behaviour 34 Results and Discussion 35 II I . Behaviour i n Laboratory Raised Fish General Methods 3 9 Observation Procedures 40 Results and Discussion 42 IV. Summary of Results 46 CHAPTER FOUR Mating Success i n Clear Water Competition I. Introduction 49 II. Background and Preliminary Studies i n Sperm Labelling 51 II I . Injection of Radioactive Tracer and Recovery of Labelled Sperm From Females... 53 IV. Stains and Autoradiographic Techniques.... 54 V. Experimental Design and Procedure 56 VI. Assessment Procedures and Sources of Error 66 VII. Results and Discussion 78 VIII. Summary of Results 90 V CHAPTER FIVE Mating Success i n Turbid Water Competition I. I n t r o d u c t i o n 92 I I . Experimental Design and Procedure 93 I I I . Results and Dis c u s s i o n 101 IV. Summary of Results < 105 CHAPTER SIX General Discussion The Adaptive S i g n i f i c a n c e of Geographic V a r i a t i o n i n the Behaviour and Morphology of P o e c i l i a  r e t i c u l a t a 107 LIERATURE CITED 122 APPENDICES A. Mean numbers of males, females and j u v e n i l e s i n 2500 cm2 areas i n the four r i v e r s 130 B. S t a t i s t i c a l values f o r B a r t l e t t ' s , ANOVA and Sch e f f e s 1 t e s t s on data from the f i e l d observations of populations i n the four r i v e r s . . 131 C. S t a t i s t i c a l values f o r B a r t l e t t ' s , ANOVA and Scheffes' t e s t s on data from the l a b o r a t o r y observations of populations from the four rivers.132 D. Mean s i z e s , and standard e r r o r s , of guppies used i n t h i s study 133 E. Formulas f o r Stains 134 vi L i s t of Tables PAGE TABLE ONE Comparison of the P h y s i c a l C h a r a c t e r i s t i c s of the Naranjo, P a r i a , Lower Aripo and Guayamare R i v e r s . . . 28 TABLE TWO Means of the Behavioural and P h y s i c a l C h a r a c t e r i s t i c s Observed and Recorded i n the Naranjo, P a r i a , Lower Aripo and Guayamare R i v e r s . . . 36 TABLE THREE Comparisons of the Means of the Behavioural and P h y s i c a l C h a r a c t e r i s t i c s Observed and Recorded i n the Naranjo, P a r i a , Lower Aripo and Guayamare R i v e r s 37 TABLE FOUR Means of the Frequencies and Durations of the Behaviours Observed and Recorded i n Laboratory Raised Males from the Naranjo, P a r i a , Lower Aripo and Guayamare R i v e r s 43 TABLE FIVE Comparison of the Means of the Frequencies and Durations of Behaviours Observed and Recorded i n Laboratory Raised Males from the Naranjo, P a r i a , Lower ari p o and Guayamare R i v e r s 44 TABLE SIX Mating Success i n Clear Water Competition -Experimental Outline (1 - 4) 61 [ TABLE SEVEN Proportions of Sperm Appearing as 'Labelled* i n Background R a d i a t i o n Control Smears... 75 TABLE EIGHT Proport i o n of L a b e l l e d Sperm i n Isotope Inj e c t e d Males 76 TABLE NINE Summary of Results of Mating Success i n Clear Water Competition, Experiments 1 - 4 81 TABLE TEN Mating Success i n Turbid Water Competition -Experimental Outline (5) 98 TABLE ELEVEN Summary of Results of Mating Success i n Turbid Water Competition, Experiment 5 104 v i i L i s t of Figures PAGE FIGURE ONE I l l u s t r a t i o n of Male Courtship Behaviours 16 FIGURE TWO Map and Location of the Island of T r i n i d a d , West Indies I n d i c a t i n g Study Rivers 21 FIGURE THREE Cages and Pond Used i n Experiments 1 - 4 59 FIGURE FOUR Outl i n e of Experimental Procedure 65 FIGURE FIVE Photomicrograph of Female Oviduct Smear I l l u s t r a t i n g an Area of Heavy Sperm Concentration Not S u i t a b l e for T a l l y i n g 6 9 FIGURE SIX Photomicrograph of Female Oviduct Smear I l l u s t r a t i n g a Low Level of L a b e l l e d Sperm 71 FIGURE SEVEN Photomicrograph of Female Oviduct Smear I l l u s t r a t i n g a High Level of L a b e l l e d Sperm 73 FIGURE EIGHT Results of Experiment 1 83 FIGURE NINE Results of Experiment 2 85 FIGURE TEN Results of Experiment 3.. 87 FIGURE ELEVEN Results of Experiment 4 89 FIGURE TWELVE Experimental Tank Used i n Experiment 5 96 FIGURE THIRTEEN Results of Experiment 5 103 FIGURE FOURTEEN The Mean Number of males (M), Females ( F ) , and J u v e n i l e s (J) i n 2500 cm2 Areas i n the Four R i v e r s 130 vi i i A C K N O W L E D G E M E N T S I w o u l d l i k e t o e x p r e s s my s i n c e r e t h a n k s t o p r o f e s s o r s N . R . L i l e y , J . D . M c P h a i l , a n d A . E . R . S i n c l a i r f o r b e i n g m e m b e r s o f my t h e s i s c o m m i t t e e a n d f o r d i s c u s s i n g t h e m a n u s c r i p t w i t h m e . I am e s p e c i a l l y i n d e b t e d t o my t h e s i s a d v i s o r D r . R o b i n L i l e y f o r p r o v i d i n g me w i t h t h e o p p o r t u n i t y t o d o t h i s r e s e a r c h a n d f o r h i s c o n t i n u e d h e l p a n d a s s i s t a n c e w h i c h h e h a s o f f e r e d t h r o u g h o u t t h e d u r a t i o n o f t h i s s t u d y . A l s o , I w o u l d l i k e t o t h a n k D r . J a c k P r i c e , D r . J . S . K e n n y , M r s . R e a n t y H o s e i n a n d M r . M a h a s e R a m l a l f o r t h e i r h o s p i t a l i t y a n d h e l p d u r i n g my s t a y i n T r i n i d a d . I am g r a t e f u l t o my w i f e M a r y a n d my m o t h e r a n d f a t h e r f o r t h e u n e n d i n g p a t i e n c e a n d s u p p o r t w h i c h t h e y h a v e p r o v i d e d o v e r t h e p e r i o d o f t h i s r e s e a r c h . T h i s w o r k w a s s u p p o r t e d b y a N a t i o n a l R e s e a r c h C o u n c i l o f C a n a d a O p e r a t i n g G r a n t t o D r . L i l e y . 1 CHAPTER ONE INTRODUCTION A major t h r u s t of e t h o l o g i c a l research has been aimed at e l u c i d a t i n g the fu n c t i o n s of the behaviour of organisms. The word f u n c t i o n i s used here not only i n the sense of 'what i s i t f o r ' but a l s o i n the stronger sense posed by Hinde (1975), "through what consequences does n a t u r a l s e l e c t i o n act to maintain the character?" These consequences, or s e l e c t i v e pressures, e l i c i t what L i l e y and Seghers (1975) r e f e r to as a species' 'response', namely i t s morphological, p h y s i o l o g i c a l and behavioural c h a r a c t e r i s t i c s . I t i s through analysing t h i s response i n terms of s u r v i v a l value, suggested Tinbergen (1965), that some suggestions can be formulated as to how the c h a r a c t e r i s t i c evolved. The guppy, P o e c i l i a r e t i c u l a t a , a l i v e bearing t e l e o s t i n the Family P o e c i l i i d a e , o f f e r s a unique opportunity to i n v e s t i g a t e the s e l e c t i v e pressures a f f e c t i n g the morphological and behavioural c h a r a c t e r i s t i c s of a speci e s . The guppy e x h i b i t s marked i n t r a s p e c i f i c v a r i a t i o n i n behaviour and morphology and e x i s t s i n a wide range of n a t u r a l environments i n the West Indies and Venezuela. Pioneering work by Winge (1922a,b; 1923, 1927, 1930, 1932, 1934), Winge and D i t l e v s o n (1938, 1948) and Haskins (1951) concentrating on sex determination and colour i n h e r i t a n c e i n the species has a l s o l e d to the establishment of a v a r i e t y of l a b o r a t o r y s t r a i n s i d e n t i f i a b l e by genetic markers. A short ovarian c y c l e (23 - 30 2 d a y s ) , r a p i d m a t u r a t i o n ( 2 - 3 m o n t h s ) , h a r d i n e s s a n d e a s e o f c o l l e c t i o n a n d s h i p m e n t c o m b i n e t o m a k e t h e g u p p y a n i d e a l o r g a n i s m t o s t u d y f r o m b o t h t h e c o m p a r a t i v e a n d e x p e r i m e n t a l a p p r o a c h e s . S t u d i e s a t t e m p t i n g t o s h e d l i g h t o n m i c r o e v o l u t i o n a r y t r e n d s i n t h e s p e c i e s h a v e c o n c e n t r a t e d o n c o r r e l a t i n g o b s e r v e d v a r i a t i o n i n b e h a v i o u r a n d m o r p h o l o g y w i t h b i o t i c a n d a b i o t i c d i f f e r e n c e s i n e n v i r o n m e n t s . T h e s e c o r r e l a t i o n s h a v e g i v e n r i s e t o h y p o t h e s e s w h i c h i n t u r n h a v e b e e n t e s t e d e x p e r i m e n t a l l y . T h i s s t u d y f o l l o w s t h i s a p p r o a c h . G u p p i e s a r e m a r k e d l y s e x u a l l y d i m o r p h i c . M a l e s e x h i b i t a n e l a b o r a t e a n d h i g h l y s p e c i e s - s p e c i f i c i n n a t e c o u r t s h i p p a t t e r n ( H a s k i n s e t a l.,1961). F e m a l e s e x h i b i t a n u m b e r , o f r e c e p t i v e b e h a v i o r p a t t e r n s w h i c h a r e u s u a l l y n e c e s s a r y f o r s u c c e s s f u l i n s e m i n a t i o n ( L i l e y , 1 9 6 6 ) . I n s i z e , t h e m a l e i s s m a l l e r t h a n t h e f e m a l e a n d w h i l e c o l o u r a t i o n i n t h e m a l e i s p r o n o u n c e d , i t i s v i r t u a l l y a b s e n t i n t h e f e m a l e . W i n g e (1922a,b, 1923, 1927, 1930, 1932, 1934,) a n d W i n g e a n d D i t e l e v s o n (1938, 1948) h a v e e x t e n s i v e l y s t u d i e d t h e s e x - d e t e r m i n i n g s y s t e m i n P . r e t i c u l a t a a s w e l l a s m o d e s o f c o l o u r p a t t e r n i n h e r i t a n c e . T h e s e a u t h o r s h a v e f o u n d s e x - d e t e r m i n a t i o n t o h a v e a g e n e t i c b a s i s i n t h e s p e c i e s , i n v o l v i n g a p a i r o f s e x - c h r o m o s o m e s a n d a n u m b e r o f s e x - g e n e s o n o t h e r c h r o m o s o m e s . T h e s y s t e m i s o f t h e X - Y t y p e w i t h a l a r g e n u m b e r o f p o l y m o r p h i c c o l o u r p a t t e r n s b e h a v i n g g e n e t i c a l l y a s t h o u g h l i n k e d t o t h e Y c h r o m o s o m e . H a s k i n s a n d H a s k i n s (1951) e x t e n d e d t h i s w o r k a n d f o u n d * a n o t h e r l a r g e c o m p l e x o f t h e p a t t e r n s a c t i n g a s t h o u g h l i n k e d t o t h e X c h r o m o s o m e a l s o . H a s k i n s e t a l . (1961) m e n t i o n e d t h a t t h e 3 system i s r e l a t i v e l y u n s p e c i a l i z e d and that the sex chromosomes are at an e a r l y stage of e v o l u t i o n a r y development as compared to D r o s o p h i l a r f o r example. As i n a l l p o e c i l i i d s , f e r t i l i z a t i o n i s i n t e r n a l . Rosen and Gorden (1953) described the f u n c t i o n a l morphology of the gonopodium, the modified anal f i n i n the male through which spermatophores are t r a n s f e r e d to the female. Sperm i s kept v i a b l e i n the f o l d s of the ovary (Winge, 1922a; S t o l k , 1950) and can be stored up to ten months (Hogarth and Sursham, 1972). One insemination may be s u f f i c i e n t to f e r t i l i z e a number of broods (Hildemann and Wagner, 1954; Baerends, Brouwer and Waterbolk, 1955). Winge (1937) suggested that new inseminations always compete s u c c e s s f u l l y with sperm already i n the ovary. Hildemann and Wagner (1954) found, however, that e a r l i e r inseminations could a l s o c o n t r i b u t e to the progeny of a given brood. The l a t t e r authors a l s o c i t e d evidence f o r m u l t i p l e inseminations i n l a b o r a t o r y stocks. Haskins, et a l . , (1961) contended that m u l t i p l e inseminations are v i r t u a l l y non-existent i n the w i l d . My research supports the f i n d i n g s of Hildemann and Wagner (see Chapter Four). Fontaine (1945) and Trinkaus and Drake (1952) have shown tha t the guppy i s a c t u a l l y ovoviviparous, the developing embryo r e c e i v i n g l i t t l e or no nourishment from the female. Breder and Rosen (1966) reviewed the research on g e s t a t i o n i n the guppy. Females e x h i b i t an ovarian c y c l e of 23 to 30 days, r i p e ova being present immediately a f t e r the release of a brood (Turner, 1937). Sexual maturity i s reached i n approximately 2 months, t h i s can vary depending upon environmental c o n d i t i o n s (Haskins 4 et a l . , 1961). Size of brood i s considered by Turner (1937), Purser (1938) and Affleck (1960). Affleck suggested that brood size i s i n part a function of female length, with numbers varying from 2 to 100. Isolated populations of guppies exist i n headstream and lowland r i v e r s on the island of Trinidad, West Indies. Headstream r i v e r s are characterized by water which i s clear, low in temperature and high i n v e l o c i t y . Predator species and numbers are low. Lowland r i v e r s , on the other hand, are slow, turbid, high in temperature and contain diverse and numerous guppy predators (Seghers, 1973; L i l e y and Seghers 1975). Correlated with headstream river conditions, L i l e y and Seghers (1975) have found populations of guppies i n which individuals are large, males are brightly coloured and which exhibit a sex r a t i o of as much as four females to one male. Guppies i n these r i v e r s c h a r a c t e r i s t i c a l l y have poorly developed anti-predator and schooling behaviours. Individuals of lowland rive r populations are smaller, less brightly coloured, exhibit a 1:1 sex r a t i o and well developed anti-predator and schooling behaviours. Of these relationships, the most extensively investigated to date i s that between predation and i t s possible sel e c t i v e effects on guppy behaviour and morphology. Seghers (1970, 1973, 1974) studied the responses of natural populations to predation. He found that populations subject to high predation pressures (such as those i n lowland rivers) were much more cohesive than those subject to predation by only a single species such as Rivulus (ty p i c a l of headstream r i v e r s ) . He 5 t e s t e d the hypothesis that schooling i s a response to predator pressure by presenting w i l d and l a b o r a t o r y r a i s e d 'schoolers' (lowland r i v e r populations) and non-schoolers (headstream r i v e r populations) i n equal numbers to l a r g e predators. He found the predators to take a greater number of 'non-schoolers' than 'schoolers'. Seghers i d e n t i f i e d d i f f e r e n c e s between populations i n r e a c t i o n d i s t a n c e , alarm thr e s h o l d and m i c r o h a b i t a t s e l e c t i o n i n the a n t i - p r e d a t o r complex of adaptions i n the species. The use of guppies r a i s e d under S i m i l a r c o n d i t i o n s i n the l a b o r a t o r y provided evidence for Segher's contention that many of the observed behavioural d i f f e r e n c e s between populations are, i n p a r t , due to genetic d i f f e r e n c e s . L i l e y and Seghers (1975) c i t e evidence to suggest that v a r i a t i o n i n s i z e may a l s o , i n p a r t , be a response to p r e d a t i o n . A headstream r i v e r predator, R i v u l u s , was found to prey s e l e c t i v e l y on small f i s h w hile l a r g e r lowland r i v e r predators were u n s e l e c t i v e or took l a r g e r guppies. Their research was based on the observed c o r r e l a t i o n between high predator d e n s i t y and s i z e and small guppies i n lowland r i v e r s and low predator density and s i z e and l a r g e guppies i n headstream r i v e r s . The suggestion t h a t predation exerted a s e l e c t i v e e f f e c t on the colour patterns of male guppies (Haskins, et a l . , 1961) has been t e s t e d by Endler (1978, 1980). Endler (1980) placed stocks c o l l e c t e d from 11 l o c a t i o n s i n T r i n i d a d randomly i n tanks representing a v a r i e t y of substrate and predator c o n d i t i o n s . A f t e r f i v e and fourteen months exposure to p r e d a t i o n , censuses of the f i s h i n the various tanks were 6 taken, although none was taken before the predators were added. The f i s h were scored f o r number, s i z e and p o s i t i o n of spots, colour and body s i z e . He found guppies to be l e s s conspicuous at high predator as compared' to low predator i n t e n s i t i e s and found a good background matching i n colour p a t t e r n s , that i s , course g r a v e l matched l a r g e spots and f i n e g r a v e l matched small spots. While Endler d i d not o f f e r s p e c i f i c d e t a i l s on h i s s c o r i n g methods, he concluded from h i s r e s u l t s t h a t n a t u r a l s e l e c t i o n acts d i f f e r e n t i a l l y on d i f f e r e n t components of colour p a t t e r n s . Furthermore he concluded that colour p a t t e r n v a r i a b i l i t y r e f l e c t s a s e l e c t i v e balance between predation and another b i o t i c f e a t u r e , sexual s e l e c t i o n . Haskins et a l . , (1961) were the f i r s t to t e s t the hypothesis that male c o l o r patterns which are b r i l l i a n t to the human eye c o n s t i t u t e a s e l e c t i v e advantage i n breeding. 'Bright* and ' d u l l ' males of inbred l a b o r a t o r y stocks were allowed to compete f o r females. Since the stocks could be i d e n t i f i e d by genetic markers, the progeny of the mating could be assessed f o r parentage. The authors found a s t a t i s t i c a l l y greater number of o f f s p r i n g to be of descent of b r i g h t males. They d i d not however, i n v e s t i g a t e the mechanism of the breeding advantage, suggesting only that i t might be through female choice, inter-male competition or a s s o c i a t e d physiology. Endler (1980) suggested on the b a s i s of h i s research that competition between males o f d i f f e r e n t colour patterns was a prime determinant of reproductive success i n low predator r i v e r s . Farr (1976) and Farr and Herrenkind (1974), on the other 7 hand, c o n t e n d t h a t b e h a v i o u r a l d i f f e r e n c e s , i n t e r m s o f v a r i a t i o n i n d i s p l a y r a t e s and a g g r e s s i o n a f f e c t i n g i n t e r m a l e c o m p e t i t i o n , were t h e p r i m e d e t e r m i n a n t s o f r e p r o d u c t i v e s u c c e s s i n t h e guppy. They s u g g e s t t h a t c o l o u r a c t s o n l y as a • c l u e * i n t e r m s o f k i n s h i p . F a r r (1977) c o n c l u d e d i n a l a t e r s t u d y t h a t f e m a l e c h o i c e b a s e d on b o t h r a r e c o l o u r ( r a r e - m a l e phenomena) and b e h a v i o u r a l c h a r a c t e r i s t i c s was t h e c e n t r a l mechanism r a t h e r t h a n i n t e r - m a l e a g g r e s s i o n . M a t i n g a number o f i n b r e d l a b o r a t o r y s t o c k s w i t h v a r i o u s b e h a v i o u r a l and m o r p h o l o g i c a l c h a r a c t e r i s t i c s , F a r r (1980) c o n c l u d e d f r o m h i s r e s u l t s t h a t h i g h c o u r t s h i p d i s p l a y r a t e i n m a l e s c o n f e r r e d a b r e e d i n g a d v a n t a g e t o m a l e s . F a r r a s s e s s e d t h e p r o g e n y o f t h e m a t i n g s and f u r t h e r c o n c l u d e d t h a t m a l e s r e s p o n d t o t h e d i s p l a y r a t e o f a c o m p e t i t o r by i n c r e a s i n g t h e i r d i s p l a y r a t e s and t h a t c o l o u r , f i r s t s u g g e s t e d by H a s k i n s e t a l . , (1961) t o be t h e a p r i m e d e t e r m i n a n t o f r e p r o d u c t i v e s u c c e s s , o n l y had e f f e c t i f t h e r e were no o v e r r i d i n g b e h a v i o u r a l d i f f e r e n c e s . [ As L i l e y and S e g h e r s (1975) have s t a t e d , i t may be / m i s l e a d i n g t o s e a r c h f o r t h e b i o l o g i c a l f u n c t i o n o f a p i e c e o f b e h a v i o u r or m o r p h o l o g y . S i m i l a r l y , i t i s m i s l e a d i n g t o s e a r c h f o r t h e p r i m e d e t e r m i n a n t o f r e p r o d u c t i v e s u c c e s s . I t i s more l i k e l y t h a t t h e i n t e r a c t i o n o f a g r e a t number o f b i o t i c and a b i o t i c f e a t u r e s d e t e r m i n e t h e s u r v i v a l v a l u e o f a c h a r a c t e r and i t s c o n t r i b u t i o n t o m a t i n g s u c c e s s . W h i l e p r e d a t i o n has been most i n t e n s i v e l y s t u d i e d i n t e r m s o f i t s s e l e c t i v e e f f e c t s on guppy b e h a v i o u r and m o r p h o l o g y , t h e r e has been a r e c e n t e f f o r t t o expand r e s e a r c h t o i n c l u d e a 8 number of a b o i t i c features, including temperature, water v e l o c i t y , and, i n t h i s study, t u r b i d i t y . L i l e y and Seghers (1975) found temperature to have an e f f e c t on the size of adult male guppies. Males of both headstream and lowland r i v e r s grew larger under a 23 °C regime than those under a 28 °C regime, although under both regimes headstream males grew larger than lowland males. There were no s i g n i f i c a n t differences i n the effects on females of the stocks used. With evidence suggesting the size differences i n males are at least i n part due to genetic differences, the authors i d e n t i f i e d another selective factor which has l i k e l y played a part i n the evolution of the species. Stream v e l o c i t y has been found to have a d i r e c t e f f e c t on the courtship behaviour of males (Crow, 1981). Increasing water ve l o c i t y causes a corresponding decrease i n courtship display . frequency, but males from headstream r i v e r s (high velocity) are better able to maintain frequency and duration of display than males from lowland r i v e r s (low v e l o c i t y ) . Using both f i s h shipped d i r e c t l y from the r i v e r s and laboratory stocks of the same populations raised under similar conditions, Crow (1981) concluded that the observed v a r i a t i o n i n a b i l i t y to maintain display has a genetic component and i s an adaptation , i n part, to stream v e l o c i t y . B a l l i n (1973) provided i n i t i a l evidence that not only does male courtship d i f f e r i n frequency and duration between d i f f e r e n t environments but he also suggested that two d i s t i n c t behavioural strategies e x i s t . In p a r t i c u l a r B a l l i n proposed that there are two routes to insemination i n males, one through 9 courtship display and active p a r t i c i p a t i o n by the female, the other involving simply a gonopodial thrust by the male made whether the female i s receptive or not. On the basis of the observed differences i n guppy morphology and the associated differences i n physical and b i o t i c conditions discussed above, and p a r t i c u l a r l y differences i n t u r b i d i t y , I predicted that males i n clear headstream rivers would rely heavily upon the v i s u a l components (displays) i n t h e i r courtship. On the other hand, I suggested that i n f i s h from turbid lowland r i v e r s , the non-visual components would play a more prominent role i n courtship (thrusting). As the two insemination strategies are present i n both types of populations, differences were expected to be quantitative rather than q u a l i t a t i v e . In headstream r i v e r s sexual selection has been thought to play a role i n the evolution of large size and bright colouration (Liley and Seghers, 1975), and i s predicted to r e s u l t i n more vigorous or persistent courtship displays, a l l features emphasizing the v i s u a l component. In lowland r i v e r s , predation i s more intense and the water i s turbid. These factors would probably re s u l t i n selection against a vigorous, conspicuous display and favour a mating strategy not as heavily r e l i a n t on v i s u a l aspects. This study was organized into two stages. In the f i r s t stage the behaviour of guppies from four populations, two from clear headstream rivers,one from a turbid lowland r i v e r and one from an intermediate midstream r i v e r , was observed i n the f i e l d and the laboratory in order to: 10 a) confirm the existence of differences i n the behaviour of males from d i f f e r e n t populations and determine whether or not they are i n the predicted d i r e c t i o n , that i s , a higher display frequency and duration i n headstream as compared to lowland males, and a higher frequency of thrusting i n lowland as compared to headstream males. It was also predicted that males from a midstream river would exhibit c h a r a c t e r i s t i c s intermediate between these two extremes. b) determine whether these differences are, i n part, due to genetic differences. Offspring of f i s h from the four populations were bred and raised under i d e n t i c a l conditions i n the laboratory and the males' courtship behaviour observed. If the differences i d e n t i f i e d i n the f i e l d persisted i n the laboratory raised f i s h , t h i s would provide strong evidence (but not proof) that the differences have a genetic basis. In the second stage of the study I attempted to test the hypothesis that the observed differences i n behaviour are to some extent adaptations to the d i f f e r e n t physical conditions, in p a r t i c u l a r to differences i n t u r b i d i t y , i n which the populations naturally occur. If the display strategy in male courtship behaviour i s an adaptation to clear water conditions, one would expect males exhibiting a high frequency and duration of display to have greater mating success i n clear water than males exhibiting lower frequencies and durations of display. Conversely, i f the thrust strategy i s an adaptation to turbid water conditions, one would expect males exhibiting high frequencies of thrusts to have greater mating success i n turbid water than males exhibiting lower frequencies of thrusts. 11 The hypothesis gave ri s e to the following predictions: (c) that headstream males (high frequency and duration of display in courtship) would have greater mating success than lowland and midstream males (lower frequencies and durations of display) when placed i n competition for females of a l l populations i n clear wa'ter. This prediction was tested i n experiments 1 and 2. (d) that lowland males (high frequency of thrusts i n courtship) would have greater mating success than headstream males (lower frequencies of thrusts) when placed i n competition for females of both populations i n turbid water. This prediction was tested i n experiment 5. Furthermore, i t would be expected that males from d i f f e r e n t populations, but exhibiting similar courtship strategies, would be equally successful at mating with females i n clear water conditions. This led to two further predictions: (e) that there would be no difference i n the mating success of lowland and midstream males when placed i n competition for females of both populations i n clear water. This prediction was tested i n experiment 3. (f) that there would be no difference i n the mating success of males from two isola t e d headstream r i v e r s when placed i n competition for females of both populations i n clear water. This prediction was tested i n experiment 4. As there was no reason to expect q u a l i t a t i v e differences i n the courtship of the males of the two d i f f e r e n t populations there was no reason to expect females to be s e l e c t i v e l y responsive to any type of courtship and therefore i t was 12 p r e d i c t e d that both headstream and lowland r i v e r females would mate f r e e l y with headstream males i n c l e a r water and lowland males i n t u r b i d water. 13 CHAPTER TWO REVIEW OF THE COURTSHIP BEHAVIOUR AND NATURAL ENVIRONMENTS OF THE GUPPY I. Behaviour The courtship behaviour of P o e c i l i a r e t i c u l a t a has been extensively studied and described. Clark and Aronson (1951) offered the f i r s t detailed description of courtship patterns and presented preliminary results on the effectiveness of these patterns i n bringing about insemination. The male pattern i s the most conspicuous, the male orients infront of the female, then arches i t s body in a sigmoid display with f u l l y open or closed caudal f i n . The male then turns and attempts to inse r t the gonopodium into the female's genital pore. While Breder and Coates (1935) suggested sexual discrimination i n the guppy to be poor, other authors including Noble and Curtis (1935) and Haskins and Haskins (1949, 1950) concluded from their studies that sexual discrimination of males for conspecific females was well developed. Breder and Coates (1935) also suggested that the females played no active role i n courtship. This contention was supported by Haskins and Haskins (1949) . Later investigations, however, supported Stepanek's (192 8) report i n which he suggested that females are acti v e l y involved. Latham (1949) elaborated on t h i s suggestion and Clark and Aronson (1951) f i n a l l y i d e n t i f i e d and described a d i s t i n c t , stereotyped female response behaviour. Kadow (1954) 14 and L i l e y (1966) o f f e r d e t a i l e d d e s c r i p t i o n s of the response which c o n s i s t s e s s e n t i a l l y of the female swimming i n a t i g h t c i r c l e w i t h the g e n i t a l pore exposed and thereby cooperating w i t h the male i n the mating attempt. B a l l i n (1973), Farr (1978) and Crow (1981) have i d e n t i f i e d s i g n i f i c a n t v a r i a t i o n s i n the c o u r t s h i p p a t t e r n of males from a number of d i f f e r e n t n a t u r a l populations. I have p r e d i c t e d t h a t males from c l e a r headstream r i v e r s w i l l e x h i b i t higher d i s p l a y frequencies (emphasizing the v i s u a l component) and males from t u r b i d lowland r i v e r s w i l l e x h i b i t higher t h r u s t frequencies (emphasizing the non-visual component). The f o l l o w i n g elements of the male's c o u r t s h i p p a t t e r n where observed and recorded. These behaviours are i l l u s t r a t e d i n Figure 1. (A) Sigmoid D i s p l a y . The sigmoid d i s p l a y begins up to 15 cm i n f r o n t of the female; the male quivers with i t s body curved i n t o and arch or an S - l i k e shape wh i l e moving from i n f r o n t of to beside the female. The male's caudal f i n i s e i t h e r f u l l y extended or t i g h t l y closed during the d i s p l a y . (B) Gonopodial Thrust. The male swims alongside and s l i g h t l y below the female, brings the gonopodium and i p s i l a t e r a l f i n forward and attempts to i n s e r t i t i n t o the female's g e n i t a l pore w i t h a quick jabbing motion. I s p e c i f i c a l l y define the gonopodial t h r u s t as a c o p u l a t i o n attempt by the male without p r i o r d i s p l a y and without p r i o r female response. (C) Copulation Attempt. I f the male's gonopodium touches the female's g e n i t a l pore f o r a f r a c t i o n of a second, a f t e r e i t h e r a sigmoid d i s p l a y or a t h r u s t , and t h i s i s 15 FIGURE 1 I l l u s t r a t i o n s of males c o u r t s h i p b e h a v i o u r s . 1 . Three v a r i a n t s of the s igmoid d i s p l a y . 2 . Gonopodia l t h r u s t . 3 . C o p u l a t i o n A t t e m p t . 4 . Gonopod ia l Swing. 5 . A g g r e s s i v e encounter ( c h a s e ) . Adapted from Baerends , Brouwer and W a t e r b o l k , 1955. See t e x t f o r f u r t h e r d e t a i l s . 16 17 followed by post-copulatory j e r k i n g i n the male (a number of je r k y jumps away from the female) the behaviour i s recorded as a c o p u l a t i o n attempt. I t would be l e s s accurate to record t h i s behaviour as an a c t u a l c o p u l a t i o n since i t i s d i f f i c u l t to e s t a b l i s h whether spermatophores have a c t u a l l y been t r a n s f e r e d during any one attempt. Evidence does e x i s t , however, to suggest that these c r i t e r i a are i n d i c a t i v e of s u c c e s s f u l inseminations. Baerends, Brouwer and Waterbolk (1955), using the oviduct smear technique, found 15 of 16 females to be inseminated a f t e r c o p u l a t i o n attempts as described above. (D) Gonopodial Swing. The gonopodial swing i s defined as the sideward and forward movement of the gonopodium by the male when there i s no female near or no attempt at insemination. Clark and Aronson (1951) and L i l e y (1966) found t h i s behaviour to increase i n frequency with increased sexual a c t i v i t y . Baerends, Brouwer and Waterbolk (1955) suggested that i t may be a displacement a c t i v i t y . (E) Aggressive Encounter. During an aggressive encounter a male gives chase or nips at another male. I f the attacked male nips or chases i n r e t u r n , t h i s i s a l s o considered as an aggressive encounter. As discussed i n the I n t r o d u c t i o n , two behavioural sequences are b e l i e v e d to r e s u l t i n insemination. In one st r a t e g y , males perform sigmoid d i s p l a y s to females and attempt to inseminate w i t h the female's cooperation (Clark and Aronson, 1951; Kadow, 1954; Baerends, Bouwer and Waterbolk, 1955; L i l e y , 1966; Farr 1975, 1980). In t h i s s t r a t e g y , which I term the d i s p l a y s t r a t e g y , the male t y p i c a l l y o r i e n t s i n f r o n t of the 18 female, e x h i b i t s a sigmoid d i s p l a y and attempts to copulate w i t h the female. Successful c o p u l a t i o n i n v o l v e s the female's cooperation, expressed as a set of stereotyped r e c e p t i v e behaviour patterns ( L i l e y , 1966). The second strategy i n v o l v e s simply the male's o r i e n t a t i o n to the female, a gonopodial t h r u s t and a c o p u l a t i o n attempt (Kadow, 1954; L i l e y , 1966; F a r r , 1980). The t h r u s t strategy does not appear to i n v o l v e female cooperation or acceptance. I t must be noted that gonopodial t h r u s t s , l i k e d i s p l a y s , r a r e l y r e s u l t i n a s u c c e s s f u l insemination and often r e s u l t i n only a small amount of sperm being t r a n s f e r e d to the female (Clark and Aronson, 1951; Baerends, Brouwer and Waterbolk, 1955; L i l e y , 1966). Farr (1980), however, contends that the frequency of gonopodial t h r u s t s r e s u l t i n g i n inseminations as compared to d i s p l a y s may be underestimated. Most research to date has used v i r g i n females, most of which immediately respond to c o u r t s h i p d i s p l a y . This biases the r e s u l t s towards insemination through d i s p l a y . Secondly, female guppies are r e c e p t i v e to male d i s p l a y s only during a 3-5 day period a f t e r the b i r t h of a brood when r i p e ova are present. During the remainder of the 30 day ovarian c y c l e females are unresponsive to male c o u r t s h i p and inseminations can only occur through t h r u s t i n g . In w i l d p o p u l a t i o n s , while the d i s p l a y strategy could r e s u l t i n more inseminations during the female's r e c e p t i v e p e r i o d , gonopodial t h r u s t i n g and sperm storage during the non-receptive period could r e s u l t i n s u c c e s s f u l f e r t i l i z a t i o n . I I . The Study Area The n a t u r a l d i s t r i b u t i o n of the guppy ranges from the c o a s t a l regions of Veneuela and B r i t i s h Guiana, through the i s l a n d s of T r i n i d a d and Tobago and i n t o both the Windward and Leeward Islands of the Lesser A n t i l l e s (Rosen and B a i l e y , (1963). Populations e x i s t i n swamps, ponds, streams and r i v e r s i n these areas, i n c l u d i n g some b r a c k i s h environments. The i s l a n d of T r i n i d a d , the general research area of t h i s study, l i e s o f f the coast of Venezuela opposite the mouth of the Orinoco R i v e r . The Northern Range, a chain of t r e e covered mountains, part of the c o a s t a l c o r d i l l e r a of Venezuela, extends along the northern margin of the i s l a n d . The average e l e v a t i o n of the range i s approximately 600-700 m ( L i l e y and Seghers, 197 5) and the peaks of E l Tucuche and E l Aripo extend above some 1000 m. A s e r i e s of short ( l e s s than 16 km), narrow, roughly p a r a l l e l r i v e r s d i s s e c t the northern slopes of the Range, emptying i n t o the Caribbean Ocean. The southern drainage of the Range i s composed of a number of s i m i l a r but longer r i v e r s which form the t r i b u t a r i e s of the l a r g e r Caroni and t o a l e s s e r extent, the Guayamare R i v e r s . These l a t t e r flow through the lowlands and empty i n t o the Gulf of P a r i a (see Figure 2 ) . While areas of low s a l i n i t y e x i s t along the northern coast and t h a t of the Gulf of P a r i a , i t i s h i g h l y probable that P.  r e t i c u l a t a does not e x i s t i n the open sea. Populations i n the northern slope r i v e r s are thus e f f e c t i v e l y i s o l a t e d from each other and from those i n southern slope and lowland r i v e r s . The 20 FIGURE 2 Map and l o c a t i o n of t h e i s l a n d o f T r i n i d a d , West I n d i e s i n d i c a t i n g s t u d y r i v e r s 1 . P a r i a R i v e r . 2 . N a r a n j o R i v e r . 3 . Lower A r i p o R i v e r . 4 . Guayamare R i v e r . A d a p t e d f r o m S e g h e r s , 1 9 7 3 . 22 upper ends of the southern r i v e r s are as e f f e c t i v e l y isolated as those i n the north but i n their lower reaches they are connected by floodwaters and t h e i r common outflow (Haskins et a l . f 1961; L i l e y and Seghers, 1975). Four r i v e r s , one on the northern slope, two on the southern slope and one i n the lowlands were selected for t h i s study. Differences i n physical and b i o t i c c h a r a c t e r i s t i c s determined the choices. The northward flowing Paria (P) (see Figure 2) i s approximately 13 km in length and i s blocked at i t s mouth by a 12 m w a t e r f a l l . The Naranjo River (also refered to as the Upper Aripo by L i l e y and Seghers, 1975) i s a tributary of the southern flowing Aripo River. The Aripo River i s punctuated at i t s midpoint by a series of 3 m high f a l l s . The major f i s h predators are iso l a t e d below the f a l l s i n the Lower Aripo. The only f i s h predator of the guppy existing i n the Paria and Naranjo Rivers i s Rivulus h a r t i i (Cyprinodontidae). Endler (197 8) contended that the freshwater prawn Macrobrachium crenulatum, which exists i n at least one of these r i v e r s , the Paria, i s a serious predator of the guppy. Endler states that the cutoff point for the eyes of arthropods i n terms of s e n s i t i v i t y i s at the orange or red wavelengths or longer. His discussion implied that t h i s may be responsible for a s h i f t i n colour patterns i n male guppies toward predominantly red patches where crenulatum i s present. My own observations of the prawn i n the Paria, based upon some 20 individuals i n the company of guppies and many more incidental encounters, have not revealed an approach or capture by the prawn. 23 Observations t h a t EJL. crenulatum i s mainly nocturnal places some question on Endler's conclusions. The t h i r d r i v e r , the Lower A r i p o , contains a number of seri o u s f i s h predators i n c l u d i n g Aequidens pulcher and C r e n i c i c h l a a l t a ( C i c h l i d a e ) , Astyanax bimaculatus. Hemibrycon sp., Hoplias malabaricus, and Hypostomus r o b i n i i ( L o r i c a r i i d a e ) (Seghers, 1973; L i l e y and Seghers, 1975; pers. obs.). Seghers (1973) i d e n t i f i e d the Lower Aripo as a midstream r i v e r , intermediate i n p h y s i c a l c h a r a c t e r i s t i c s between headstream and lowland r i v e r s . The f o u r t h study r i v e r , the Guayamare, flows through low l y i n g a g r i c u l t u r a l land and empties i n t o the Gulf of P a r i a v i a the Caroni Swamp. The Guayamare dra i n s to some extent i n t o the Caroni R i v e r , the major drainage f o r the southern slopes, v i a an a r t i f i c i a l canal l o c a t e d some 16 km east of the Caroni Swamp. The predator species i n the Guayamare River are much the same as the Lower Aripo with the a d d i t i o n of two f u r t h e r species of characids. Seghers (1973) and L i l e y and Seghers (1975) provide f u r t h e r and more s p e c i f i c d e t a i l s on the predator fauna of these and other T r i n i d a d r i v e r s . These authors a l s o presented data on va r i o u s a b i o t i c c h a r a c t e r i s t i c s of the r i v e r s . The data suggested the f o l l o w i n g trends from headstream r i v e r s (the Naranjo and Paria) through midstream (the Lower Aripo) to lowland R i v e r s (the Guayamare); i n c r e a s i n g width, depth, volume of flow, temperature and t u r b i d i t y and decreasing v e l o c i t y and shade. Since my p r e d i c t i o n s concerning d i f f e r e n c e s i n behaviour of populations of guppies i n these r i v e r s were based on these trends, 24 e s p e c i a l l y i n t u r b i d i t y , i t was e s s e n t i a l t o examine whether the t r e n d s remained c o n s i s t a n t over t i m e . The f o l l o w i n g s e c t i o n p r e s e n t s my a n a l y s i s o f the p h y s i c a l c h a r a c t e r i s t s o f the f o u r r i v e r s u t i l i z e d i n t h i s s t u d y . Methods One l o c a t i o n on each of the f o u r r i v e r s , the N a r a n j o , the P a r i a , the Lower A r i p o and the Guayamare was s e l e c t e d and i d e n t i f i e d as r e p r e s e n t a t i v e of t h a t r i v e r . U s i n g t o p o g r a p h i c a l maps, a number of l o c a t i o n s were v i s u a l l y examined (where v e g e t a t i o n a l l o w e d ) and a g e n e r a l a r e a was chosen w h i c h was a c c e s s i b l e , c o n t a i n e d p o p u l a t i o n s of guppies and was r e p r e s e n t a t i v e of the u n i f o r m c o n d i t i o n s i n each r i v e r . A g r i d r e f e r e n c e map was made of each l o c a t i o n on which was i n d i c a t e d t h e p h y s i c a l appearance of the r i v e r a t t h a t l o c a t i o n . S p e c i f i c p o i n t s were i d e n t i f i e d a t each l o c a t i o n u s i n g a tape measure and a random number t a b l e . These p o i n t s were i d e n t i f i e d on the g r i d r e f e r e n c e map i n c e n t i m e t e r s from permanant landmarks. R e c o r d i n g s were always made a t the s p e c i f i c p o i n t s i n d i c a t e d on the map. The l o c a t i o n s were each v i s i t e d on 8 days d u r i n g t h e p e r i o d May t o November, 197 8 f o r the e x p r e s s purpose of c o l l e c t i n g p h y s i c a l d a t a . A l l . t h e r i v e r s were v i s i t e d on any one day and the v i s i t s were spaced 20 t o 30 days a p a r t . The or d e r i n whi c h the r i v e r s were v i s i t e d on any one day was r o t a t e d . T h i s approach was used i n or d e r t o i n c o r p o r a t e any s e a s o n a l e f f e c t s on the r i v e r s d u r i n g t h e study p e r i o d and a t the same time c o n t r o l f o r s h o r t term weather changes. 25 The following physical c h a r a c t e r i s t i c s were measured; 1. Depth: i n cm at 3 points i n each of the Naranjo (N), Paria (P), and Lower Aripo (LA) and at 2 points i n the Guayamare (G). 2. Temperature: i n C at the same points i n each of N, Pf LA, and G. 3. Stream v e l o c i t y : using a meter st i c k , a stopwatch and a small buoyant fla s k , the time i t took the flask to travel 1 m was recorded. Three replicates were taken at the same points i n the N, P, LA, and G. Data was recorded i n m/sec. 4. Turbidity: a) using a sechi disk, the depth at which the disk disappeared from sight was recorded i n cm. Recordings were made at 2 of the 3 points i n the N, P, and LA, and the 2 points i n the G. b) using a t u r b i d i t y meter. The meter was composed of a 30 cm grey polyethylene cylinder 7.5 cm i n diameter and sealed at one end with a disk of clear polyethylene. This formed the container for a column of water. This cylinder was placed, once f i l l e d with water, atop a black polethylene box with one open side. In the top of the box was a 2 cm aperature l i n e d on the inside with black foam rubber. The l i g h t receptor of a Lunasix 3 Light Meter f i t t e d into t h i s aperture and was shielded from a l l l i g h t save that coming through the column of water. A l i g h t was then placed over the top of the cylinder (powered by a water protected and f u l l y recharged 5 v o l t 26 battery). This device was designed by N.R. L i l e y to measure r e l a t i v e rather than absolute values of t u r b i d i t y . The Lunasix 3 Light Meter was set at din 16 and readings were taken on a scale 0 - 30, 0 being the lowest l i g h t l e v e l and therefore r e f l e c t -ing the highest t u r b i d i t y . Control readings were taken with the cylinder empty. Since these read i n a l l cases 10.3 on the scale, the actual scale readings were used as data. One column of water was measured at each of 2 points i n the N, P, LA, and G. 5. Light S t r i k i n g the Surface: was measured with a Lunasix 3 Light Meter held at the water's surface and set at din 19. The scale on the meter offered a r e l a t i v e measure (15.0 = approx. 2800 lux, 22.0= approx. 350000 lu x ) . Three replicate measures were taken at 10 m i n t e r v a l s at both the middle and edge of the N, P, LA, and G locations. Results The means and standard errors of the f i v e physical c h a r a c t e r i s t i c s measured i n the four r i v e r s are summarized i n Table 1. Depth and l i g h t reaching the surface were found to increase from the headstream r i v e r s to the lowland r i v e r . Water v e l o c i t y was found to decrease from headstreams to lowlands with the exception of the Paria, which was r e l a t i v e l y slow in comparison to^ the Naranjo but s t i l l faster than the lowland Guayamare. Temperature increased from headstream to lowland 27 although the trend was not found to be as d i s t i n c t as the others. T u r b i d i t y decreased markedly from lowland to headstream. The trends i d e n t i f i e d i n the p h y s i c a l c h a r a c t e r i s t i c s of the four r i v e r s i n 1978 are g e n e r a l l y c o n s i s t e n t w i t h those i d e n t i f i e d by Seghers (1973) and L i l e y and Seghers (1975). On the b a s i s of these r e s u l t s I p r e d i c t e d that male P. r e t i c u l a t a i n the c l e a r Naranjo and P a r i a r i v e r s would e x h i b i t a higher frequency of d i s p l a y strategy than would males from the t u r b i d Guayamare R i v e r . I a l s o made the p r e d i c t i o n that Guayamare males would r e l y more h e a v i l y on the non-visual c o u r t s h i p strategy ( t h r u s t i n g ) than would headstream males and that males from the Lower Aripo would occupy a midpoint between the two extremes. TABLE 1. Comparison of the physical characteristics of the Naranjo, Paria, Lower Aripo and Guayamare Rivers. Characteristic Mean Depth (cm) Mean Temperature (°C) Mean Water Velocity (m/sec) Mean Turbidity Sechi Disk (cm) Turbidity meter*5 Mean Light Reaching0 River Surface RIVER PARIA LOWER ARIPO 30.71 (2.863) 44.31 (1.897) 24.21 (0.099) 25.89 (0.160) 0.143 (0.007) 0.162 (0.009) NARANJO 30.27 (0.831) 3 25.48 (0.103) 0.646 (0.738) 100.0 (0.000) 9.91 (0.149) 15.62 (0.026) 100.0 (0.000) 10.03 (0.033) 16.56 (0.424) 31.27 (3.840) 8.03 (0.328) 18.51 (0.308) GUAYAMARE 114.24 (1.430) 25.69 (0.159) 0.047 (0.004) 5.400 (0.465) 5.04 (0.124) 20.78 (0.491) a Standard error in brackets. b Relative measure based on 0 (turbid) - 10.3 (clear) scale. See text for detai ls , c Relative measure based on 0 (low) - 30 (high) scale. See text for detai l s . 29 CHAPTER THREE GEOGRAPHIC VARIATION IN THE COURTSHIP BEHAVIOUR OF MALE P o e c i l i a r e t i c u l a t a I . I n t r o d u c t i o n The o b s e r v a t i o n s o f p o p u l a t i o n s o f g u p p i e s i n t h e i r n a t u r a l e n v i r o n m e n t s r e p o r t e d i n t h i s c h a p t e r were c a r r i e d o u t f o r two r e a s o n s . The f i r s t was t o c o n f i r m t h e r e p o r t by S e g h e r s ( 1 9 7 8 ) , b a s e d on l a b o r a t o r y o b s e r v a t i o n s , t h a t g u p p i e s f r o m p r e d a t o r d e n s e , t u r b i d l o w l a n d r i v e r s w o u l d e x h i b i t a h i g h e r d e g r e e o f c o h e s i o n and l i v e c l o s e r t o s h o r e t h a n g u p p i e s i n c l e a r , low p r e d a t o r h e a d s t r e a m r i v e r s . S e g h e r s (1978) i n t e r p r e t e d t h i s i n c r e a s e d c o h e s i o n and p r o x i m i t y t o s h o r e i n l o w l a n d r i v e r s a s r e s p o n s e s t o p r e d a t i o n p r e s s u r e . G r e a t e r c o h e s i o n i n l o w l a n d r i v e r s c a n a l s o be p r e d i c t e d on t h e b a s i s o f t h e g r e a t e r t u r b i d i t y o f t h e s e r i v e r s . L o c a t i o n o f mates i n t h e n a r r ow, c l e a r h e a d s t r e a m r i v e r s w o u l d a p p e a r t o be much l e s s a p r o b l e m t h a n i n t h e w i d e , t u r b i d l o w l a n d r i v e r s . T h e r e f o r e I p r e d i c t e d a h i g h e r l e v e l o f c o h e s i o n i n t h e Guayamare R i v e r (G) t h a n i n t h e h e a d s t r e a m N a r a n j o (N) and P a r i a (P) R i v e r s , w i t h t h e Lower A r i p o R i v e r (LA) r e f l e c t i n g i t s m i d p o i n t t u r b i d i t y ( s e e C h a p t e r 2) between t h e two e x t r e m e s . I a l s o p r e d i c t e d t h a t G and LA f i s h w o u l d o c c u p y a p o s i t i o n i n t h e r i v e r c l o s e r t o s h o r e t h a n t h o s e i n t h e N and P. The s e c o n d a i m o f t h e o b s e r v a t i o n s o f n a t u r a l p o p u l a t i o n s 30 i n the f i e l d was to t e s t the p r e d i c t i o n t h a t male guppies i n t u r b i d lowland r i v e r s would more f r e q u e n t l y e x h i b i t the t h r u s t s t r a t e g y i n t h e i r c o u r t s h i p whereas those i n headstreams would more f r e q u e n t l y e x h i b i t the v i s u a l o r i e n t e d d i s p l a y s t r a t e g y . I t was p r e d i c t e d t h a t N and P males would e x h i b i t (1) a higher frequency of sigmoid d i s p l a y s , (2) a g r e a t e r d u r a t i o n i n sigmoid d i s p l a y s and (3) a lower frequency of gonopodial t h r u s t s than males from the LA or G. No d i f f e r e n c e s were p r e d i c t e d i n the number of c o p u l a t i o n attempts. I f d i f f e r e n c e s as d e s c r i b e d above were found, the q u e s t i o n arose whether the d i f f e r e n c e s i n behaviour were a r e s u l t of e x p e r i e n t i a l i n f l u e n c e s during development or whether they had a g e n e t i c b a s i s . To d i s t i n g u i s h between the two p o s s i b l i t i e s , f i s h from the four p o p u l a t i o n s were r a i s e d under i d e n t i c a l c o n d i t i o n s i n the l a b o r a t o r y . As the f i s h matured, t h e i r behaviour was examined, again under standard l a b o r a t o r y c o n d i t i o n s . I f the b e h a v i o u r a l d i f f e r e n c e s between p o p u l a t i o n s p e r s i s t e d under these c o n d i t i o n s i t would p r o v i d e strong support f o r the c l a i m (but not proof) t h a t the d i f f e r e n c e s have a g e n e t i c b a s i s . I n v e s t i g a t i o n s of the p o p u l a t i o n s i n t h e i r n a t u r a l environments i s the s u b j e c t of the f i r s t s e c t i o n of t h i s chapter. The a n a l y s i s of behaviour i n l a b o r a t o r y r a i s e d f i s h i s the s u b j e c t of the second s e c t i o n . 31 I I . Behaviour i n the Natural Environments Methods The studies discussed below were carried out during the period May to November, 1978. Preliminary observations of the Naranjo, Paria and Lower Aripo Rivers up to 1 km upstream and downstream from the locations i d e n t i f i e d i n Chapter Two showed that guppies occurred i n a number of d i s t i n c t microhabitats within each r i v e r . In the N and P Rivers these microhabitats were of three types. The gli d e was defined as an area of r e l a t i v e l y shallow water (approximately 1 - 15 cm deep) flowing at a r e l a t i v e l y high v e l o c i t y . Glides were found at both the edges and i n the middle of the two r i v e r s . A shallow was an area of deeper (approximately 15 - 40 cm) and slower water. Shallows were found only along the edges of the r i v e r s . Pools were r e l a t i v e l y slow moving bodies of water (at times no recordable velocity) with a minimum depth of 40 cm and averaging 100 cm i n the N and 81.5 cm in the P. In the LA guppies were present i n both shallows and pools but were not seen i n glides. The Guayamare River presented a somewhat d i f f e r e n t s i t u a t i o n . Guppies were found to be thinly d i s t r i b u t e d within 1 km of the location descibed i n Chapter 2. Since a number of other locations on the G were easily accessible, i t was decided to observe guppies i n two further locations i n order to provide a comparable sample. The G does not contain g l i d e s , shallows or pools as described above. There are two discernible 32 m i c r o h a b i t a t s i n which guppies occur, one along the edge of the r e l a t i v e l y uniform main body of the r i v e r , the other i n l a r g e , slow moving pools. While depth does not vary appreciably between the two m i c r o h a b i t a t s , water v e l o c i t y i s n o t i c a b l y reduced i n the pools (0.08 m/sec i n the main body of the r i v e r against 0.005 m/sec i n the p o o l s ) . In order to obtain measures of behaviour r e p r e s e n t a t i v e of guppies occuring i n the e n t i r e r i v e r s , groups of guppies were observed according to the f o l l o w i n g schedule; Naranjo: 8 g l i d e s , 8 shallows, 4 pools = 20 l o c a t i o n s P a r i a : 8 g l i d e s , 8 shallows, 5 pools = 21 l o c a t i o n s Lower A r i p o : 8 shallows, 8 pools = 16 l o c a t i o n s Guayamare: 8 main body, 3 pools = 11 l o c a t i o n s I had hoped to observe f i s h i n each of 8 microh a b i t a t s i n each r i v e r but the r e l a t i v e infrequency of some and the r e s t r i c t e d access to others brought t h i s number down. I r e f e r to my observations t a k i n g place on groups of f i s h rather than schools i n order to avoid any confusion with respect to the connotations a s s o c i a t e d with the l a t t e r . Schools defined as cl o s e aggregates of f i s h with some measure of i n t e g r i t y were found only to e x i s t i n the main body of the G and to some extent i n the shallows and pools of the LA. Guppies i n the N and P, while not being d i s t r i b u t e d randomly throughout the r i v e r s , could not be s a i d to e x i s t i n i d e n t i f i a b l e aggregates. The f o l l o w i n g p h y s i c a l and behavioural c h a r a c t e r i s t i c s were observed and recorded i n each of the 6 8 l o c a t i o n s . 33 A. General behavioural and p h y s i c a l parameters. 1. Number of guppies per 2500 cm2: A 2500 cm.2 g r i d with markings along the edges at every cm was gently lowered i n t o the water and placed on the bottom substrate of g l i d e s and shallows i n the N f P, and LA R i v e r s ; 5 minutes were allowed before any observations were c a r r i e d out. The number of guppies i n the g r i d were counted every minute f o r 3 minutes noting number of a d u l t males, females, and j u v e n i l e s . In the G r i v e r and the pools of the N f P f and LA t u r b i d i t y and/or depth obscured the g r i d so i n these l o c a t i o n s i t was held approximately 30 cm above the water surface and numbers per minute f o r 3 minutes were recorded on a tape recorder and l a t e r t r a n s c r i b e d i n t o a f i e l d notebook. 2. Distance between f i s h : Using the g r i d and the recording methods described above, the distance between two f i s h at the edge of the g r i d was recorded i n cm. S t a r t i n g i n the bottom r i g h t corner of the g r i d , 15 distances between separate p a i r s of f i s h were recorded, moving i n a clockwise d i r e c t i o n around the g r i d . A mean was then c a l c u l a t e d f o r each l o c a t i o n . 3. Depth: The depth of the water at each l o c a t i o n was measured i n cm by lowering a tape measure i n the center of the g r i d . 4. Distance to shore: the distance of a group of f i s h to shore was measured using a tape measure, distance to shore being defined here as the distance between the 34 edge of the r i v e r at the w a t e r l i n e and the nearest guppy. F i v e measurements were taken at each l o c a t i o n , spaced 1 minute apart, and a mean c a l c u l a t e d . 5. V e l o c i t y of water: A f t e r a l l the observations had been completed, i n c l u d i n g those one male c o u r t s h i p described below, water v e l o c i t y was measured using the method described i n Chapter 2. A mean was c a l c u l a t e d over three r e p l i c a t e s . I t should be pointed out t h a t , as mentioned e a r l i e r , f i s h i n the LA and to a greater extent the G tend to be i n small schools, with the re s t of the r i v e r s being devoid of guppies. The measure of number of guppies per 2500 cm2 i n these r i v e r s t h e r e f o r e does not accurately represent f i s h d e n s i t y since i t measures only the numbers a s s o c i a t e d with schools. The measure more accurately represents the cohesion of f i s h i n areas where they are found i n the r i v e r s . B. Male Courtship Behaviours. The f o l l o w i n g procedures were used i n recording the co u r t s h i p behaviours of males. A male was i d e n t i f i e d i n the general g r i d area (holding the g r i d was dispensed with at t h i s point) and v i s u a l l y followed f o r three minutes. Observations of males which disappeared from s i g h t w i t h i n three minutes were disregarded. For each of f i v e males at each l o c a t i o n the f o l l o w i n g behaviours were recorded: 1. Number of sigmoid d i s p l a y s per 3 minutes. 2. Number of gonopodial t h r u s t s per 3 minutes. 3. Number of co p u l a t i o n attempts per 3 minutes. Durations of sigmoid d i s p l a y s were also recorded using a 35 stopwatch a f t e r the above had been completed. Ten durations were u s u a l l y recorded per l o c a t i o n , t h i s was reduced to f i v e i n some due to the infrequency of d i s p l a y and/or low density of f i s h . A maximum of 2 durations were recorded f o r any one male. S t a t i s t i c a l A n a l y s i s A l l data were transformed to log(x+l) i n order to ensure normality and homogeneity of variance (Quenouille, 1950). A l l s t a t i s t i c a l analyses were c a r r i e d out on the transformed data. Adjusted means were then obtained using the adjustment recommended by E l l i o t (1973); 1.15 times the variance of the transformed counts was added to the mean of the transformed counts and the a n t i l o g of t h i s adjusted mean was then taken and 1 subtracted i n order to obta i n the adjusted mean. Homogeneity of variance was confirmed using B a r t l e t t ' s Test (Snedecor and Cochran, 1967) at the 0.01 l e v e l of s i g n i f i c a n c e (see Appendix B). The data were then subjected to a n a l y s i s of vari a n c e . Those comparisons i n which the ANOVA i d e n t i f i e d s i g n i f i c a n t d i f f e r e n c e s were then subjected to Scheffe*s (1959) Test f o r s p e c i f i c comparisons between r i v e r s . The r e s u l t s of the ANOVAs and Scheffe Tests are summarized i n Table 3. The F values, degrees of freedom and l e v e l s of s i g n i f i c a n c e f o r the ANOVAs and the Scheffe F s t a t i s t i c s , c o n t r a s t values and l e v e l s of s i g n i f i c a n c e are included i n Appendix B. Results Table 2 l i s t s the means of the behavioural and p h y s i c a l c h a r a c t e r i s t i c s observed and recorded i n the four r i v e r s . Table 3 i n d i c a t e s a number of s i g n i f i c a n t d i f f e r e n c e s between these 36 TABLE 2. Means of the behavioural and physical characteristics observed and recorded in the Naranjo, Paria, Lower Aripo and Guayamare Rivers. Characteristic NARANJO PARIA tSSS GUAYAMARE Number of fish per 2500 cn/ Distance between fish (cm) Distance to shore (cm) Water velocity (m/sec) River ARIPO 2.81 5.63 6.95 12.35 27.99 18.61 7.73 4.12 Depth of water 3 5 n 3 2 A Q ^ 8 3 > 1 5 49.26 71.04 30.36 16.86 0.477 0.142 0.059 0.070 Number of sigmoid 3 6 2 5 4 Q , 4 3 . g . displays/3 min l - m Number of gonopodial n „ 7 n 7 f i , 9 9 9 Q ~ thrusts/3 min u > 4 / U , / C ) Number of copulation attempts/3 mm Duration of sigmoid . 1 0 . n o 0 7 0 0 0 - , J ^ „ T , W 4.13 4.08 2.73 2.87 display (sec) 37 TABLE 3. Comparisons of the means of the behavioural and physical characteristics observed and recorded in the Naranjo, Paria, Lower Aripo and Guayamare Rivers. Characteristic ANOVA Scheffe Comparisons G-LA G-N G-P LA-N LA-P N-P Number of fish , n n n n i ^ , n a P v M ^ n , A „ M b per 2500 cm^ Distance between fish (cm) < 0.0001 G>LA G>N G>P LA>N - P?N < 0.0001 - N>G P>G N>LA P>LA Depth of water Q Q Q 0 7 G>LA G>N G>P Distance to shore (cm) 0.0071 - N>G P?G - P>LA P?[ fm%lcT0CUy ° - 0 0 0 2 " N > G " N?LA Number of sigmoid < 0.0001 - N>G P>G N>LA P>LA -di splays/3 mm Number o f gonopodial < 0 . 0 0 0 1 G>LA G>N G>P -thrusts/3 min Number of copulation 0 473? c attempts/3 min. ' Duration of sigmoid < _ p > . display (sec) a Scheffe'comparisons significant at the 0.01 level of significance. b Scheffe'comparisons not significant at the 0.01 level of significance. c ANOVA not significant at the 0.01 level of significance. Scheffe'not performed. 38 c h a r a c t e r i s t i c s . Keeping i n mind that number of f i s h per 2500 cm.2 i s not a true measure of density across the entire LA and G r i v e r s , the measure does indicate than G guppies are s i g n i f i c a n t l y more cohesive i n s p e c i f i c areas than guppies i n the N, P, or LA. Both LA and P f i s h e x i b i t greater numbers per area than those i n the N. The distance between f i s h was also s i g n i f i c a n t l y lower i n both the G and LA than i t was i n the N and P. These results tend to support the prediction, based on similar findings by Crow (1981) and Seghers (1974) in laboratory observations, that guppies i n lowland r i v e r s l i v e i n more cohesive units and i n greater proximity to eachother than do those i n headstream r i v e r s . Fish i n the high predator G stay closer to shore and exist i n areas of greater depth than do those i n the low predator N and P. This supports Segher's (1973) contention that remaining close to shore i s , i n part, a response to increased predation. Both LA and N f i s h remained closer to shore than P f i s h . The analysis of male courtship behaviour revealed a number of s i g n i f i c a n t differences between populations, a l l in the predicted d i r e c t i o n s . Males from the headstream N and P r i v e r s exhibited s i g n i f i c a n t l y more sigmoid displays and displays were of s i g n i f i c a n t l y longer duration than those of males i n the LA and G. Conversely, males i n the G were found to exhibit a s i g n i f i c a n t l y higher rate of gonopodial thrusting than those i n the other three r i v e r s . The low number of copulations observed i n the four r i v e r s did not d i f f e r s i g n i f i c a n t l y . The differences i n courtship i d e n t i f i e d where consistent with those found i n the same stocks raised i n the laboratory 39 ( B a l l i n , 1973; Snyder, 1978). Crow (1981), studying the P a r i a and Caparo (lowland r i v e r ) populations under l a b o r a t o r y c o n d i t i o n s obtained s i m i l a r f i n d i n g s . He found P a r i a males to e x i b i t s i g n i f i c a n t l y more d i s p l a y s than Caparo males while the l a t t e r e x h i b i t e d s i g n i f i c a n t l y more t h r u s t s than the former. The three p r e d i c t i o n s ; t h a t N and P males would e x i b i t (1) a higher frequency of sigmoid d i s p l a y s , (2) a greater d u r a t i o n i n sigmoid d i s p l a y s and (3) a lower frequency of t h r u s t s than males from the LA and G, were supported. Also as p r e d i c t e d , f i s h from the G e x h i b i t e d a higher l e v e l of cohesion than those of the N and P. The midstream LA population was found to occupy and intermediate p o s i t i o n f o r t h i s c h a r a c t e r i s t i c . The number of observed c o p u l a t i o n attempts were low and no s i g n i f i c a n t d i f f e r e n c e s were i d e n t i f i e d . These f i n d i n g s support the hypothesis t h a t w h i l e a l l populations e x i b i t both t h r u s t s and d i s p l a y s , males from c l e a r headstream populations r e l y more on the v i s u a l l y o r i e n t e d d i s p l a y strategy i n t h e i r c o u r t s h i p w h i l e males from t u r b i d lowland populations r e l y more on the t h r u s t s t r a t e g y . I I I . Behaviour i n Laboratory Raised F i s h Methods Stocks were c o l l e c t e d from Naranjo, P a r i a , Lower Aripo and Guayamare R i v e r s and kept i n separate g l a s s tanks of 21 and 38 1 capacity at the Asa Wright Nature Centre (Simla), T r i n i d a d . These f i s h w i l l henceforth be r e f e r e d to as the p a r e n t a l  generation. The stock tanks were f i l l e d and r e g u l a r l y changed 40 w i t h t a p w a t e r , t h e s o u r c e o f w h i c h was a n a t u r a l s t r e a m . They were m a i n t a i n e d i n n a t u r a l l i g h t and t e m p e r a t u r e c o n d i t i o n s i n a l a r g e open a i r room a t t h e C e n t r e . Young o f a l l f o u r s t o c k s were c o l l e c t e d as t h e y a p p e a r e d and were p l a c e d i n s e p a r a t e t a n k s , and a r e r e f e r e d t o as t h e  f i r s t g e n e r a t i o n . The f i s h i n t h e s e t a n k s were a l l o w e d t o m a t u r e and were u s e d as s u b j e c t s o f t h e b e h a v i o u r a l a n a l y s i s , w h i c h was begun 2 months a f t e r t h e e s t a b l i s h m e n t o f t h e f i r s t g e n e r a t i o n . F o o d f o r a l l s t o c k s c o n s i s t e d o f t h e c o m m e r c i a l l y p r e p a r e d d r i e d f o o d T e t r a - m i n o c c a s i o n a l l y s u p p l e m e n t e d w i t h l o c a l l y c o l l e c t e d m o s q u i t o l a r v a e . B e h a v i o u r a l o b s e r v a t i o n s were c a r r i e d o u t i n a s e p a r a t e room i n t h e C e n t r e between 800 and 1700 h o u r s . One t a n k o f 21 1 c a p a c i t y was u t i l i z e d as t h e o b s e r v a t i o n t a n k . The t a n k was s h i e l d e d on t h r e e s i d e s and i l l u m i n a t e d by a f l u o r e s c e n t l i g h t s u s p e n d e d 2 m above t h e t a n k . O b s e r v a t i o n P r o c e d u r e Ten m a t u r e f i r s t g e n e r a t i o n f e m a l e s o f a g i v e n s t o c k were t a k e n f r o m t h e i r t a n k and p l a c e d i n t h e o b s e r v a t i o n t a n k . Ten f i r s t g e n e r a t i o n m a l e s o f t h e same p o p u l a t i o n were t h e n t a k e n f r o m t h e t a n k and p l a c e d i n a 10 1 c a p a c i t y t a n k . A d r a w i n g was made of e a c h male i n d i c a t i n g i t s c o l o u r p a t t e r n t o be u s e d f o r s u b s e q u e n t i d e n t i f i c a t i o n d u r i n g t h e o b s e r v a t i o n s . The m a l e s were t h e n p l a c e d w i t h f e m a l e s i n t h e o b s e r v a t i o n t a n k a l e f t f o r a 30 m i n u t e a c c l i m a t i z a t i o n p e r i o d . Each male was t h e n o b s e r v e d f o r 10 m i n u t e s d u r i n g w h i c h b e h a v i o u r s were r e c o r d e d u s i n g a R u s t r a k r e c o r d e r c o n n e c t e d t o a f o u r key b o a r d . 41 The f o l l o w i n g behaviours were recorded f o r each male: 1. Number of sigmoid d i s p l a y s per 10 minutes. 2. Number of gonopodial t h r u s t s per 10 minutes. 3. Number of c o p u l a t i o n attempts per 10 minutes. 4. Durations of sigmoid d i s p l a y s (by keeping the key i n (1.) depressed f o r the duration of the d i s p l a y ) . 5. Number of gonopodial swings per 10 minutes. 6. Number of aggressive encounters per 10 minutes (due to the infrequency of c o p u l a t i o n attempts and aggressive encounters, the same key was used f o r these behaviours, one stroke i n d i c a t i n g the former, two r a p i d s t r o k e s , the l a t t e r . D e s c r i p t i o n s and d e f i n i t i o n s of these behaviours are presented i n Chapter 2. Observing and recording these behaviours i n 10 males f o r 10 minutes per male c o n s t i t u t e d one t r i a l . Three t r i a l s were c a r r i e d out f o r each of the four populations. A f t e r each t r i a l the f i s h were anaesthetised i n 1:600 MS 222 (Tricane methane sulphonate - Sandoz) to d i s t i l l e d water. The mean s i z e s of a l l f i s h used i n observations and experiments are recorded i n Appendix D. The data was subjected to the same s t a t i s t i c a l a n a l y s i s as that used and described i n the previous s e c t i o n . Homogeneity of variance was comfired using B a r t l e t t ' s Test (Snedecor and Cochran, 1967) at the 0.01 l e v e l of s i g n i f i c a n c e . F values, degrees of freedom and l e v e l s of s i g n i f i c a n c e f o r the ANOVAs and the Scheffe F s t a t i s t i c s , c o n t r a s t values and l e v e l s of s i g n i f i c a n c e are included i n Appendix C. 42 R e s u l t s The means of the f r e q u e n c i e s and d u r a t i o n s o f the b e h a v i o u r s o b s e r v e d and r e c o r d e d a r e shown i n T a b l e 4. T a b l e 5 summarizes t h e r e s u l t s of the s t a t i s t i c a l a n a l y s e s . L a b o r a t o r y r a i s e d N a r a n j o and P a r i a males d i s p l a y e d a t h i g h e r f r e q u e n c i e s and f o r l o n g e r d u r a t i o n s t h a n Guayamare males. G males i n t u r n performed g o n o p o d i a l t h r u s t s a t h i g h e r f r e q u e n c i e s than b o t h N and P males. These r e s u l t s a r e c o n s i s t e n t w i t h those observed i n the f i e l d , namely t h a t headstream males e x i b i t a h i g h e r f r e q u e n c y of the d i s p l a y s t r a t e g y and l o w l a n d males a h i g h e r f r e q u e n c y of the t h r u s t s t r a t e g y i n t h e i r c o u r t s h i p . S i n c e the f i r s t g e n e r a t i o n f i s h were r a i s e d under i d e n t i c a l l a b o r a t o r y c o n d i t i o n s away from n a t u r a l s e l e c t i v e a g e n t s , the f i n d i n g s p r o v i d e s t r o n g e v i d e n c e t h a t t h e r e i s a g e n e t i c c o n t r i b u t i o n t o t h e observed d i f f e r e n c e s . D i f f e r e n c e s between the f i e l d ( T a ble 3) and l a b o r a t o r y ( T a b l e 5) r e s u l t s a r e almost c o n f i n e d t o the midstream Lower A r i p o R i v e r . The s i g n i f i c a n t l y g r e a t e r f r e q u e n c y of t h r u s t i n g e x h i b i t e d by G males as opposed t o LA males i d e n t i f i e d i n the f i e l d was not c o n f i r m e d i n the l a b o r a t o r y r a i s e d f i s h . L a b o r a t o r y r a i s e d LA males were found, however, t o e x i b i t a s t a t i s t i c a l l y g r e a t e r number of d i s p l a y s t h a n G males, a d i f f e r e n c e not found i n the f i e l d . S i m i l a r l y , LA males e x h i b i t e d a h i g h e r f r e q u e n c y of t h r u s t i n g t h a n P males i n t h e l a b o r a t o r y . Whether these d i f f e r e n c e s between f i e l d and l a b o r a t o r y a r e a r e s u l t of e x p e r i e n t i a l f a c t o r s d u r i n g growth, d i f f e r e n c e s between the o b s e r v a t i o n tank and the n a t u r a l streams or d i f f e r e n c e s i n my methods of r e c o r d i n g o b s e r v a t i o n s 43 TABLE 4. Means of the frequencies and durations of the behaviours observed and recorded i n laboratory raised males from the Naranjo, Paria, Lower Aripo and Guayamare Rivers. Behaviour NARANJO River PARIA LOWER ARIPO GUAYAMARE Number of sigmoid displays/10 min Number of gonopodial thrusts/10 min Number of copulation attempts/10 min Duration of sigmoid display (sec) Number, of gonopodial swings/10 min Number of aggressive encounters/TO min 1.4.20 . 3.57 0.23 2.63 16.21 1.42 11.07 2.60. 0.13 2.09 8.71 1.70 4.71 5.81 0.10 1.64 8.70 0.03 2.57 9.03 0.13 1,26 10.27 1.87 44 TABLE 5. Comparison of the means of the frequencies and durations of behaviours observed and recorded i n laboratory raised males from the Naranjo, Paria, Lower Aripo and Guayamare Rivers. Behaviour ANOVA Scheffe''comparisons G-LA G-N G-P LA-N LA-P N-P Number of sigmoid <0.0001 LA>G.a' .N.>G P>G N>LA P/LA - b di splays/10 mm Number of gonopodial n n n n , r > N r > p , fl>p thrusts/10 min < 0.0001 - G>N G>P LA>P -Number of Copulation . c attempts/10 min U . W D D - - - -d i s r a p U y n ( s L ) i 9 m ° i d < 0 - 0 0 0 1 LA>G N>G P>G N>LA P>LA N>P Number of gonopodial y n n m . . . . . , . ; , V r , swings/10 min ; < 0 . 0 0 0 1 - N>G - N>LA. - N>P Number of aggressive , n 7 1 c . c encounters/10 min i . u / i o a Scheffe comparisons significant at the 0.0.1 level of significance, b Scheffe'''comparisons not significant, at the 0.01 level of significance. c ANOVA not significant at the 0.01 level of significance. Scheffe^not performed. 45 i s d i f f i c u l t to e s t a b l i s h . It i s inte r e s t i n g to note, however, that a l l these differences involve the LA stock and generally support i t s status as an intermediate population between the headstreams and lowland. As i n the f i e l d , the number of copulation attempts observed was low and there were no s i g n i f i c a n t differences between the populations. Two behaviours were measured i n the laboratory and not i n the f i e l d : gonopodial swings because they were d i f f i c u l t to distinguish i n the f i e l d s i t u a t i o n and aggressive encounters because they were non-existant i n the f i e l d . While two other laboratory studies found P males to be s i g n i f i c a n t l y more aggressive than the lowland G ( B a l l i n , 1973) and Caparo (Crow, 1981) males, no s i g n i f i c a n t differences were found i n t h i s study. Farr (1975) also found aggression to be v i r t u a l l y non-existent i n the f i e l d and found i t to decrease reproductive success i n laboratory strains of guppies (Farr, 1980). These findings shed some doubt on B a l l i n ' s (1973) suggestion that aggression i s functional in inter-male competition and Gandolfi's (1971) and Gorlick's (1976) contentions that i t i s important i n establishing status and s o c i a l hierarchies i n wild populations. N males were found to exhibit s i g n i f i c a n t l y more gonopodial swings than males of the other populations. The significance of gonopodial swings i n terms of male courtship has yet to be determined. Clark and Aronson (1951) and L i l e y (1966) have found t h i s behaviour to increase with increased sexual a c t i v i t y , and Crow (1981) speculated that i t may 46 f u n c t i o n as an ' i n t e n t i o n movement' before d i s p l a y . B a l l i n (1973) and Snyder (197 8) found gonopodial swings to be i n greater frequency i n lowland populations as opposed to headstreams. Crow (1981) has suggested t h a t these observations may r e f l e c t non-courtship gonopodial swings which represent displacement a c t i v i t i e s as f i r s t suggested by Baerends, Brouwer and Waterbolk (1955). IV. Summary of Results Four populations of P o e c i l i a r e t i c u l a t a , two from c l e a r headstream r i v e r s (Naranjo and P a r i a ) , one from a t u r b i d lowland r i v e r (Guayamare) and another from an intermediate midstream r i v e r (Lower Aripo) were observed i n t h e i r n a t u r a l environments and i n the l a b o r a t o r y and a number of behaviours recorded. A number of p r e d i c t i o n s were t e s t e d through observation. A. F i e l d Observations. 1. The lowland population was found to be more cohesive than the headstream pop u l a t i o n s . 2. The lowland population was found to i n h a b i t areas c l o s e r to shore than the headstream pop u l a t i o n s . 3. Males of the lowland population were found to e x h i b i t a higher frequency of gonopodial t h r u s t s during c o u r t s h i p than headstream males. 4. Males of the headstream populations where found to e x h i b i t a higher frequency and duration of sigmoid d i s p l a y s during t h e i r c o u r t s h i p than lowland males. 47 5. No differences were found i n the frequency of copulation attempts exhibited by males of the four populations. 6. Behaviours performed by f i s h from the midstream LA population were generally found to be intermediate between the headstreams and lowland. B. Laboratory observations. 1. With f i s h from the. four populations born raised under i d e n t i c a l conditions i n the laboratory, males of the lowland population were found to exhibit a higher frequency of gonopodial thrusts during courtship than headstream males. 2. Males of the headstream populations were found to exhibit a higher frequency and duration of sigmoid displays during t h e i r courtship than lowland males. 3. Males from one headstream river (N) were found to perform a higher frequency of gonopodial swings than males from the other populations. 4. No differences were found i n the frequency of copulation attempts or aggressive encounters between males of the four populations. 5. Differences between f i e l d and laboratory results tended to support the intermediate character of the midstream (LA) population. The results support the prediction that males from clear headstream r i v e r s rely more heavily on the v i s u a l display strategy i n t h e i r courtship, while males from the turbid lowland rive r rely more heavily on the non-visual thrust strategy i n t h e i r courtship. Since the differences were found pe r s i s t i n stocks bred and raised under i d e n t i c a l conditions i n 48 t h e l a b o r a t o r y , s t r o n g e v i d e n c e was found t o s u p p o r t the p r e d i c t i o n t h a t the d i f f e r e n c e s a r e , a t l e a s t i n p a r t , g e n e t i c a l l y d e t e r m i n e d . 49 C H A P T E R F O U R M A T I N G S U C C E S S O F M A L E S I N C O M P E T I T I O N F O R F E M A L E S I N C L E A R W A T E R I . I n t r o d u c t i o n O b s e r v a t i o n s o f g u p p i e s i n f o u r r i v e r s i n T r i n i d a d h a v e e s t a b l i s h e d t h a t s i g n i f i c a n t d i f f e r e n c e s e x i s t b e t w e e n p o p u l a t i o n s i n l e v e l s o f c o h e s i o n a n d m a l e c o u r t s h i p b e h a v i o u r s . S t r o n g e v i d e n c e w a s f o u n d t o s u g g e s t t h a t t h e d i f f e r e n c e s h a v e , i n p a r t , a g e n e t i c b a s i s . T h i s i m p l i e s t h a t t h e d i f f e r e n t b e h a v i o u r s a r e t h e p r o d u c t o f n a t u r a l s e l e c t i o n a n d t h u s h a v e s o m e a d a p t i v e s i g n i f i c a n c e f o r t h e f i s h i n t h e d i f f e r e n t e n v i r o n m e n t s . I n o r d e r t o b e a d a p t i v e , t h e b e h a v i o u r a l p a t t e r n s m u s t m a k e t h e o r g a n i s m b e t t e r a b l e t o s u r v i v e a n d r e p r o d u c e a s c o m p a r e d t o o t h e r m e m b e r s o f t h e s a m e s p e c i e s ( W i l s o n , 1 9 7 5 ) . T h e y m u s t , i n o t h e r w o r d s , c o n f e r s o m e m e a s u r e o f r e p r o d u c t i v e s u c c e s s o n t h e o r g a n i s m s . E x p e r i m e n t s d e s c r i b e d i n t h i s c h a p t e r a n d c h a p t e r 5 w e r e d e s i g n e d t o t e s t t h e h y p o t h e s i s t h a t d i f f e r e n c e s i n c o h e s i o n a n d m a t i n g s t r a t e g y e x h i b i t e d b y t h e p o p u l a t i o n s a r e a d a p t a t i o n s , i n p a r t , t o d i f f e r e n c e s i n t h e t u r b i d i t y o f t h e r i v e r s i n w h i c h t h e p o p u l a t i o n s n a t u r a l l y o c c u r . M a l e s f r o m t h e f o u r p o p u l a t i o n s w e r e p l a c e d i n c o m p e t i t i o n f o r m a t i n g w i t h f e m a l e s u n d e r c l e a r ( t h i s c h a p t e r ) a n d t u r b i d ( C h a p t e r 5) w a t e r c o n d i t i o n s . M a t i n g s u c c e s s w a s d e t e r m i n e d o n t h e b a s i s o f n u m b e r o f i n s e m i n a t i o n s a n d s p e r m c o n t r i b u t e d t o f e m a l e s b y 50 m a l e s o f t h e d i f f e r e n t p o p u l a t i o n s . S p e r m w e r e i d e n t i f i e d u s i n g a r a d i o i s o t o p e l a b e l l i n g t e c h n i q u e a n d s t a n d a r d a u t o r a d i o g r a p h i c m e t h o d s . T h e h y p o t h e s i s l e d t o t h e f o l l o w i n g p r e d i c t i o n s f o r m a t i n g s u c c e s s i n c l e a r w a t e r : 1. T h a t m a l e s f r o m t h e c l e a r h e a d s t r e a m p o p u l a t i o n s ( N a r a n j o a n d P a r i a ) w o u l d b e m o r e s u c c e s s f u l a t m a t i n g , t h a n m a l e s f r o m t h e t u r b i d m i d s t r e a m ( L o w e r A r i p o ) a n d l o w l a n d ( G u a y a m a r e ) r i v e r s , w h e n p l a c e d i n c o m p e t i t i o n f o r f e m a l e s o f a l l p o p u l a t i o n s . A s s u m i n g t h a t c o l o u r a t i o n a n d d i s p l a y e v o l v e d u n d e r e p i g a m i c s e x u a l s e l e c t i o n i n b o t h t y p e s o f e n v i r o n e m e n t , t h e p r e s s u r e o f t h i s t y p e o f s e l e c t i o n w o u l d b e e x p e c t e d t o b e m o r e i n t e n s e a n d p r o g r e s s e d f u r t h e r i n c l e a r w a t e r . I f t h e v i g o r o u s a n d c o n s p i c u o u s d i s p l a y s t r a t e g y i n h e a d s t r e a m m a l e s i s a r e s p o n s e t o t h i s p r e s s u r e , i t s h o u l d b e m o r e l i k e l y t o a t t r a c t a n d s t i m u l a t e a l l f e m a l e s . T h i s p r e d i c t i o n w a s t e s t e d i n t w o e x p e r i m e n t s : E x p e r i m e n t 1 : H e a d s t r e a m ( N a r a n j o ) m a l e s i n c o m p e t i t i o n f o r m a t i n g w i t h l o w l a n d m a l e s ( G u a y a m a r e ) f o r f e m a l e s o f b o t h p o p u l a t i o n s . E x p e r i m e n t 2 : H e a d s t r e a m (N) m a l e s i n c o m p e t i t i o n f o r m a t i n g w i t h m i d s t r e a m m a l e s f o r f e m a l e s o f b o t h p o p u l a t i o n s . 2. T h a t t h e r e w o u l d b e n o s i g n i f i c a n t d i f f e r e n c e s i n m a t i n g s u c c e s s b e t w e e n m a l e s o f t h e m i d s t r e a m ( L A ) a n d l o w l a n d (G) r i v e r s w h e n p l a c e d i n c o m p e t i t i o n f o r f e m a l e s o f b o t h p o p u l a t i o n s . W h i l e L A m a l e s e x h i b i t e d b e h a v i o u r s i n t e r m e d i a t e b e t w e e n t h o s e e x h i b i t e d b y h e a d s t r e a m a n d l o w l a n d m a l e s , t h e 51 courtship of LA males tended to be more similar to that of lowland males. Also, while the water of the headstream r i v e r s was clear, the lowland and midstream ri v e r s d i f f e r e d only in the degree of t u r b i d i t y . I therefore predicted that there would be no differences i n mating success between males of the lowland and midstream populations. This prediction was tested i n one experiment: Experiment 3 : Midstream (LA) males placed i n competition for mating with lowland males (G) for females of both populations. 3. That there would be no s i g n i f i c a n t differences i n mating success between males of the two headstream populations (N and P) when placed i n competition for females of both populations. Since no major differences i n courtship behaviour exist between the two populations and the environmental conditions i n which they exist are similar, no differences i n mating success were expected between males of these populations. This prediction was tested i n one experiment: Experiment 4 : Headstream (N) males placed i n competition for mating with headstream males (P) for females of both populations. II . Background and Preliminary Studies i n Sperm Labelling. The radioisotopes thymidine - 3H and thymidine 14c are exclusively incorporated into the DNA of c e l l s (Reichard and Esteborn, 1951). These isotopes have been used extensively in studying DNA synthesis and the c e l l cycle i n man (eg. B e l l , et 52 a l . f 1967; Cole and Mckalen, 1963), the r a t ( S a s k i , 1965), mice (Layde and Baserga, 1964), Drosophila ( P l a u t , et a l . , 1966) and a number of other organisms and c e l l c u l t u r e s (see Baserga and Malamud, 1969, for an extensive review). The p o s s i b i l i t y of using these isotopes i n l a b e l l i n g the sperm of P o e c i l i a r e t i c u l a t a was f i r s t suggested to me by N.R. L i l e y . Later we became aware of s t u d i e s by B i l l a r d (1966) and Crowley (196 8) documenting s u c c e s s f u l i n c o r p o r a t i o n of thymidine - 3H i n t o guppy sperm using standard autoradiographic methods. I c a r r i e d out a number of p i l o t s tudies i n 1977 - 1978 i n order to determine i f the isotopes could be used with success. The p r e l i m i n a r y s t u d i e s , using standard autoradiographic procedures (see Gross, et a l . , 1951; J o f t e s , 1963; Rogers, 1967; and Baserga and Malamud, 1969, for comprehensive reviews ) , revealed that from 70 - 80% of sperm taken from males 21-28 days a f t e r an i n j e c t i o n of the isotopes could be l a b e l l e d . A spermatazoa was defined as l a b e l l e d i f i t had incorporated the isotope i n t o i t s DNA. This i n c o r p o r a t i o n was i d e n t i f i e d by a black g r a i n i n the emulsion over the c e l l evident upon microscopic examination (see Figures 5 - 7 ) . I t was a l s o found that l a b e l l e d sperm could be s u c c e s s f u l l y recovered from females mated with i n j e c t e d males fo r a period of seven days. Neither the i n j e c t i o n not the isotope were found to have a s i g n i f i c a n t e f f e c t on the c o u r t s h i p behaviours of isotope i n j e c t e d males as compared to s a l i n e i n j e c t e d and non-injected males. 53 I I I . I n j e c t i o n o f R a d i o a c t i v e T r a c e r and R e c o v e r y o f L a b e l l e d Sperm From F e m a l e s . I n j e c t i o n T e c h n i q u e s - M a l e s M a l e s were t a k e n f r o m p a r e n t a l s t o c k t a n k s and a n a e s t h e t i z e d one a t a t i m e i n a p e t r i d i s h c o n t a i n i n g 1:600 MS 222 ( T r i c a n e methane s u l p h o n a t e - Sandoz) t o d i s t i l l e d w a t e r . The a n a e s t h e t i z e d male was p l a c e d on a g l a s s s l i d e , t h e gonopodium l a y e d f o r w a r d and t h e a b d o m i n a l r e g i o n s t r o k e d w i t h a b l u n t p r o b e . T h i s e f f e c t i v e l y s t r i p p e d t h e male o f sperm. The male was t h e n p l a c e d under a d i s s e c t i n g m i c r o s c o p e on a wet c l o t h s o a k e d i n s a l i n e s o l u t i o n . Two u l o f t h y m i d i n e ( m e t h y l - 14c) - o f 1.0 cu / 1 c o n c e n t r a t i o n was drawn i n t o a 50u\L c a p a c i t y H a m i l t o n s y r i n g e f i t t e d w i t h a #30 n e e d l e . The n e e d l e was s l i d a n t e r i o r l y i n t o t h e a b d o m i n a l r e g i o n o f t h e m a l e , j u s t d o r s a l t o t h e gonopodium. The i s o t o p e was i n j e c t e d i n t o t h e f i s h . As t h e n e e d l e was w i t h d r a w n , t h e wound was h e l d c l o s e d w i t h a p a i r o f f o r c e p s i n o r d e r t o e n s u r e m i n i m a l l o s s o f i s o t o p e . Where s a l i n e i n j e c t e d m a l e s were u s e d , t h e s t r i p p i n g and i n j e c t i n g p r o c e d u r e s were t h e same , e x c e p t t h a t a d i f f e r e n t s y r i n g e was u s e d and 2.0 u^l o f s a l i n e (0.6% NaCl) s o l u t i o n i n j e c t e d . A f t e r t h e i n j e c t i o n a f i s h was a l l o w e d t o r e c o v e r i n a p e t r i d i s h c o n t a i n i n g d i s t i l l e d w a t e r . F i s h were t h e n i s o l a t e d f r o m f e m a l e s and o t h e r e x p e r i m e n t a l g r o u p s i n t a n k s c o n t a i n i n g t a p w a t e r ( f r o m a l o c a l f r e s h w a t e r stream) w i t h 0.5 gm/1 m e t h y l e n e b l u e d i s s o l v e d . 54 S a m p l i n g T e c h n i q u e s - F e m a l e s F e m a l e s w e r e t a k e n f r o m t h e e x p e r i m e n t a l c a g e s ( s e e E x p e r i m e n t a l D e s i g n , t h i s c h a p t e r ) , a n a e s t h e t i z e d i n MS 2 2 2 , a n d p l a c e d u n d e r a d i s s e c t i n g m i c r o s c o p e o n a w e t c l o t h s o a k e d i n s a l i n e s o l u t i o n . M i c r o p i p e t t e s f a s h i o n e d b y f l a m i n g a n d p u l l i n g 2 . 5 mm d i a m e t e r g l a s s t u b i n g w e r e u s e d t o t a k e o v i d u c t s a m p l e s f r o m t h e f e m a l e s . T h e m i c r o p i p e t t e w a s f i r s t p a r t i a l l y f i l l e d w i t h d i s t i l l e d w a t e r a n d t h e n i n s e r t e d i n t o t h e f e m a l e ' s g e n i t a l o p e n i n g . T h e d i s t i l l e d w a t e r w a s f l u s h e d 4 t i m e s f r o m p i p e t t e t o o v i d u c t t o p i p e t t e . T h e m i c r o p i p e t t e w a s t h e n w i t h d r a w n a n d t h e c o n t e n t s f l u s h e d o n t o a s t e r i l i z e d g l a s s s l i d e . T h e p r o c e d u r e w a s r e p e a t e d t h r e e t i m e s f o r e a c h f e m a l e . T h e s a m p l e w a s s p r e a d o v e r a p p r o x i m a t e l y t w o - t h i r d s o f t h e s l i d e u s i n g t h e ' b l o o d s m e a r t e c h n i q u e ' . T h e s m e a r s w e r e t h e n a l l o w e d t o a i r d r y . A new m i c r o p i p e t t e w a s u s e d f o r e a c h f e m a l e . I V . S t a i n s a n d A u t o r a d i o g r a p h i c T e c h n i q u e s . S t a i n i n g T h e s l i d e s w i t h d r y s m e a r s , e a c h r e p r e s e n t i n g a n o v i d u c t s a m p l e f r o m a f e m a l e o r a s p e r m s a m p l e f r o m a m a l e , w e r e p l a c e d i n t h e r e m o v a b l e t r a y s o f s t a i n i n g d i s h e s a n d s t a i n e d a s f o l l o w s ; 1 . S o a k e d i n 3 c h a n g e s o f d i s t i l l e d w a t e r , 1 0 m i n u t e s e a c h . 2 . S t a i n e d i n M a y e r ' s h e m a t o x y l i n f o r 3 m i n u t e s ( s e e A p p e n d i x E f o r f o r m u l a ) . 55 3. Rinsed i n d i s t i l l e d water. 4. Blued i n 1.0% sodium acetate, 5 minutes. 5. Rinsed i n d i s t i l l e d water. 6. Stained with aqueous eosin for 1 minute (see Appendix E for formula) . 7. Rinsed i n d i s t i l l e d water and allowed to dry. 8. One passage 50% ethanol for 5 minutes, then 2 passes 95% ethanol, 5 minutes each and allowed to dry. A l l reagents were maintained at 18 °C i n a c o l d water bath during the s t a i n i n g procedure and the hematoxylin replaced r e g u l a r l y . Autoradiographic Techniques The method of coating the smears with l i q u i d emulsion i s o u t l i n e d i n Baserga and Malamud (1969). The e n t i r e procedure subsequently to be discussed was c a r r i e d out i n a dark room under a l i g h t f i t t e d with a Kodak Wratten S e r i e s I (red) f i l t e r . A 120 ml b o t t l e of Kodak NTB - 3 Nuclear Emulsion was taken from r e f r i g e r a t e d storage and the emulsion melted i n the b o t t l e i n a water bath at 43.0+ 0.5 °C. S i x t y ml of the emulsion was d i l u t e d with 60 ml d i s t i l l e d water i n a beaker and s t i r r e d slowly with a g l a s s rod. A l l bubbles were removed from the d i l u t e d emulsion. Each s l i d e was dipped i n the emulsion f o r 2 seconds, ensuring that the e n t i r e s l i d e was coated. The s l i d e s were then placed v e r t i c a l l y on the edge of paper t o w e l l i n g and allowed to dry. The f i l t e r e d l i g h t was turned o f f during the drying p e r i o d . 56 Once dry, the s l i d e s were placed i n s l i d e boxes taped with Scotch Brand black e l e c t r i c a l tape. Each box contained a small amount of anhydrous CaS04 ( D r i e r i t e ) wrapped i n gauze. The boxes were sealed with black tape and stored at room temperature for 14 days. A f t e r the 14 day exposure period s l i d e s were removed from t h e i r boxes and processed as f o l l o w s : 1. Kodak D19 developer f o r 3 minutes. 2. D i s t i l l e d water for 1 minute. 3. Kodak F i x e r for 10 minutes. 4. 1:15 d i l u t i o n of Edwal Hypoeliminator to d i s t i l l e d water f o r 1 minute and allowed to dry. V. Experimental Design and Procedure A l l f i s h used i n the competitive mating experiments were c o l l e c t e d from the four r i v e r s , the Naranjo (N), P a r i a (P), Lower Aripo (LA) and Guayamare (G) and held i n the p a r e n t a l stock tanks described i n Chapter 3. Only f i s h c o l l e c t e d d i r e c t l y from the r i v e r s were used i n these experiments. A l l experiments were conducted i n an outdoor concrete pool at the Asa Wright Nature Centre (Simla), T r i n i d a d . The pool measured 270 cm X 200 cm X 43 cm (Figure 3 ) . A continuous flow of c l e a r water was fed i n t o the pool from a l o c a l n a t u r a l stream. The water l e v e l i n the pool was maintained at 24 cm depth. Each of the two r e p l i c a t e s per experiment (see below) were conducted i n one of the 6 cages placed i n the pool. The cages were constructed by L i l e y and myself out of wood and 57 p l a s t i c m e s h w h i c h d i d n o t a l l o w t h e e s c a p e o f f i s h f i n c l u d i n g n e w b o r n ( F i g u r e 3 ) . A l i d w h i c h f i t t e d s e c u r e l y o n t h e t o p o f t h e c a g e a l l o w e d a c c e s s . W a t e r t e m p e r a t u r e o v e r t h e e x p e r i m e n t a l p e r i o d , A u g u s t t o O c t o b e r , 1 9 7 8 , a v e r a g e d 26.3 ° C a t 1 0 0 0 h o u r s a n d 26.2 ° C a t 1 6 0 0 h o u r s . W a t e r w a s m a i n t a i n e d i n c l e a r c o n d i t i o n a n d d e b r i s c o l l e c t i n g o n o r a r o u n d t h e c a g e s w a s r e m o v e d r e g u l a r l y . F i s h w e r e f e d o n t h e c o m m e r c i a l l y p r e p a r e d d r i e d f o o d T e t r a - M i n o c c a s s i o n a l l y s u p p l e m e n t e d w i t h l o c a l l y c o l l e c t e d m o s q u i t o l a r v a e . F o u r c o m p e t i t i v e m a t i n g e x p e r i m e n t s w e r e c o n d u c t e d i n c l e a r w a t e r . I n e a c h e x p e r i m e n t , m a l e s o f t w o o f t h e f o u r p o p u l a t i o n s , N , P , L A a n d G , w e r e p l a c e d i n c o m p e t i t i o n f o r f e m a l e s o f t h e s a m e p o p u l a t i o n s . D e t a i l s o f t h e f o u r e x p e r i m e n t s a r e s u m m a r i z e d i n T a b l e 6. T w o r e p l i c a t e s w e r e c o n d u c t e d w i t h i n e a c h e x p e r i m e n t w i t h t h e i s o t o p e i n j e c t e d p o p u l a t i o n o f m a l e s r e v e r s e d . T h i s w a s d o n e t o c a n c e l o u t a n y e f f e c t s t h e i s o t o p e m i g h t h a v e o n t h e m a l e s . I t a l s o c o m p e n s a t e d f o r t h e f a c t t h a t n o t a l l s p e r m i n i s o t o p e i n j e c t e d m a l e s c o u l d b e e x p e c t t o b e l a b e l l e d ( s e e S e c t i o n V I , t h i s c h a p t e r ) . I n e x p e r i m e n t 1, r e p l i c a t e 1, N a r a n j o m a l e s i n j e c t e d w i t h i s o t o p e w e r e p l a c e d i n c o m p e t i t i o n w i t h G u a y a m a r e m a l e s i n j e c t e d w i t h s a l i n e s o l u t i o n . I n r e p l i c a t e 2, G u a y a m a r e m a l e s i n j e c t e d w i t h i s o t o p e w e r e p l a c e d i n c o m p e t i t i o n f o r m a t i n g w i t h N a r a n j o m a l e s i n j e c t e d w i t h s a l i n e s o l u t i o n . T h e d a t a f r o m t h e s e t w o r e p l i c a t e s w a s l a t e r c o m b i n e d f o r a n a l y s i s . E x p e r i m e n t 2 i n c l u d e d t w o r e p l i c a t e s o f L A a n d N m a l e s p l a c e d 58 F I G U R E 3 C a g e s a n d p o o l u s e d i n E x p e r i m e n t s 1 -A . D i a g r a m a t i c o r g a n i z a t i o n o f e x p e r -i m e n t a l c a g e s i n p o n d . 1. D r a i n 2. W a t e r i n f l o w B . P r o p o r t i o n s o f a n e x p e r i m e n t a l c a g e . 3. R e m o v a b l e l i d 4. F i n e g u a g e p l a s t i c m e s h 5. W a t e r l e v e l m a i n t a i n e d i n p o n d 59 270 CM CAGE 1 CAGE 2 CAGE 3 T • s:.' o i — i CAGE 4 CAGE 5 CAGE 6 o o CNJ H 122 CM — ~ — | 60 i n c o m p e t i t i o n f o r m a t i n g , e x p e r i m e n t 3, t w o r e p l i c a t e s o f L A a n d G m a l e s a n d e x p e r i m e n t 4, t w o r e p l i c a t e s o f N a n d P m a l e s ( s e e T a b l e 6 ) . T h e f o l l o w i n g i s a d e s c r i p t i o n o f t h e e x p e r i m e n t a l m e t h o d u s e d i n a l l t h e r e p l i c a t e s f o r a l l t h e e x p e r i m e n t s . F o r t h e s a k e o f b r e v i t y a n d c l a r i t y , t h e f i r s t r e p l i c a t e o f e x p e r i m e n t 1 i s u s e d a n e x a m p l e . T h e m e t h o d i s i l l u s t r a t e d i n F i g u r e 4. T w e n t y f i v e m a l e s w e r e r e m o v e d f r o m t h e N s t o c k t a n k a n d w e r e s t r i p p e d a n d i n j e c t e d w i t h 2.0 i^ l o f t h y m i d i n e - 14c a s d e s c r i b e d i n s e c t i o n I I I . T h i r t y m a l e s f r o m t h e G s t o c k w e r e t h e n s t r i p p e d a n d i n j e c t e d w i t h 2.0 t i l s a l i n e s o l u t i o n . T h e m a l e s f r o m t h e t w o p o p u l a t i o n s w e r e t h e n p l a c e d i n s e p a r a t e h o l d i n g t a n k s . T h i s c o n s t i t u t e d d a y 0 o f r e p l i c a t e 1, e x p e r i m e n t 1 ( o n t h e s a m e d a y r e p l i c a t e 2 w a s b e g u n , w i t h t h e i n j e c t i o n r e v e r s e d ) . O n d a y 5 o f t h e e x p e r i m e n t t h i s e n t i r e s t r i p p i n g a n d i n j e c t i o n p r o c e d u r e w a s r e p e a t e d . O n d a y 10, 35 N f e m a l e s a n d 35 G f e m a l e s w e r e r e m o v e d f r o m t h e i r r e s p e c t i v e s t o c k t a n k s a n d c l i p p e d f o r l a t e r i d e n t i f i c a t i o n . T h i s i n v o l v e d c l i p p i n g o f f t h e 2 p o s t e r i o r r a y s (6,7) o f t h e a n a l f i n o f N f e m a l e s a n d t h e 2 a n t e r i o r r a y s (1,2) o f G f e m a l e s . W h i l e t h e r a y s w e r e f o u n d t o r e g e n e r a t e , t h e c l i p p i n g p r o c e d u r e w a s s u f f i c i e n t t o p o s i t i v e l y i d e n t i f y t h e f e m a l e s o v e r t h e e x p e r i m e n t a l p e r i o d . A t t h i s t i m e t h e f e m a l e s ' o v i d u c t s w e r e f l u s h e d u s i n g a m i c r o p i p e t t e a n d s a l i n e s o l u t i o n i n o r d e r t o r e m o v e a n y s p e r m w h i c h m i g h t b e p r e s e n t . T h e f e m a l e s w e r e t h e n p l a c e d t o g e t h e r , i s o l a t e d f r o m t h e m a l e s , i n o u t d o o r c a g e 1 ( F i g u r e 3 ) . T h e s a m e p r o c e d u r e w a s c a r r i e d TABLE 6. Mating Success in Clear Water Competition Experimental Outline (1 - 4) EXPERIMENT POPULATION TYPE COMPETING POPULATIONS REPLICATE FEMALE POPULATION FEMALES MATED ANAL RAYS CLIPPED MALE POPULATION MALES MATED 4 Isolated Head-stream Lowland Mid-stream Head-stream Mid-stream - Lowland Head-stream N G N G LA 30 30 30 30 30 LA N LA G 30 30 30 30 30 LA G 6,7/1,2 1,2/6,7 1,2 / 6,7 6,7/1,2 6,7 / 1,2 LA* N LA N* LA* Head-stream Naranjo - Guayamare Lower Aripo - Naranjo Lower Aripo - Guayamare Naranjo - Paria 1 2 1 2 1 2 1 2 N 30 30 30 30 30 30 1,2/6,7 6,7/1,2 1,2/6,7 LA G* N* P N P* 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 CAGE a indicates male population injected with isotope. 62 out for females for replicate 2, except the rays clipped were reversed and the females placed i n cage 2. The cages were checked regularly for young and these removed. On day 24 females were removed from the outdoor cages and the cages cleared of any debris and young. Thirty females from each stock were i d e n t i f i e d under the dissecting microscope and replaced i n their o r i g i n a l cages. The remaining females, included i n case of m o r t a l i t i e s , were discarded i n another outdoor pond not used i n t h i s study. On day 25, 20 N males and 20 G males were added to the 30 females from each stock already present i n cage 1. This constituted the beginning of the mating period. The remaining 5 isotope injected N males were held i n the holding tank. Sperm samples from these males taken on day 28 and 5 other N males taken from the cage at the end of the experiment were used to obtain an accurate assessment of the proportion of sperm l a b e l l e d at the middle and end of the mating period (Figure 4, c ) . Smears taken from these males w i l l henceforth be referred to as l a b e l l e v e l control smears. The remaining 10 saline injected G males were held i n the i r holding tank for stripping on day 28 (Figure 4, B). Sperm smears from these males served as controls i n order to measure background radiation (naturally occurring radiation) and emulsion contamination by l i g h t . Both of these factors cause black grains to appear i n the emulsion. If the grains occur over spermatazoa, they cannot be distinguished from grains caused by isotope i n the DNA. Smears taken from these males w i l l henceforth be refered to as background radiation control 63 s m e a r s . A f t e r t h e s e m a l e s w e r e s t r i p p e d a n d s m e a r s p r e p a r e d , t h e y w e r e m e a s u r e d u s i n g c a l i p e r s , t h e n k i l l e d i n a h i g h c o n c e n t r a t i o n o f MS 222, a n d p r e s e r v e d i n f o r m a l d e h y d e . O n d a y 32 t h e m a t i n g p e r i o d o f t h e e x p e r i m e n t w a s t e r m i n a t e d ( F i g u r e 4 A ) . T h e f e m a l e s w e r e s a m p l e d a n d o v i d u c t s m e a r s p r e p a r e d a s d e s c r i b e d i n s e c t i o n I I I , 5 N m a l e s s t r i p p e d a n d l a b e l l e v e l c o n t r o l s m e a r s p r e p a r e d , a l l f i s h m e a s u r e d u s i n g c a l i p e r s , k i l l e d i n a h i g h c o n c e n t r a t i o n o f MS 222, a n d p r e s e r v e d i n f o r m a l d e h y d e . A s e a c h s m e a r w a s m a d e , i t w a s i d e n t i f i e d u s i n g a d i a m o n d e t c h i n g p e n . T h e s m e a r s w e r e t h e n s t a i n e d a n d f i x e d a s d e s c r i b e d i n s e c t i o n V I a n d t h e n s t o r e d i n s l i d e b o x e s w i t h t e n b a c k g r o u n d r a d i a t i o n c o n t r o l s m e a r s d i s t r i b u t e d a m o n g s t t h e m . A l l s m e a r s w h e r e l a t e r s u b j e c t e d t o f u r t h e r a u t o r a d i o g r a p h i c p r o c e s s i n g a s d e s c r i b e d i n s e c t i o n I V . P r o c e d u r e s f o r t h e o t h e r t h r e e e x p e r i m e n t s , L A - N , L A - G a n d N - P , a n d t h e r e p l i c a t e s t h e r e i n w e r e i d e n t i c a l t o t h o s e d e s c r i b e d a b o v e . E a c h r e p l i c a t e w a s c a r r i e d o u t i n a s e p a r a t e c a g e , t h e t w o r e p l i c a t e s o f a n y o n e e x p e r i m e n t b e i n g s t a r t e d o n t h e s a m e d a y . T h e e x p e r i m e n t s t h e m s e l v e s w e r e s t a g g e r e d d u e t o a v a i l a b l e w o r k s p a c e a n d t h e a m o u n t o f w o r k i n v o l v e d i n a n y o n e p r o c e s s i n g d a y . T h e s e t t i n g o f t h e m a t i n g p e r i o d a t 7 d a y s w a s a t r a d e o f f b e t w e e n t w o f a c t o r s , t h e p e r i o d o v e r w h i c h h i g h l e v e l s o f l a b e l l e d s p e r m c o u l d b e e x p e c t e d t o b e p r e s e n t i n i s o t o p e i n j e c t e d m a l e s a n d t h e p e r i o d o f t i m e a f t e r i n s e m i n a t i o n o v e r w h i c h s p e r m w o u l d r e m a i n a c c e s s i b l e b y o v i d u c t f l u s h i n g . My p i l o t d a t a s u g g e s t e d t h a t t h e m a x i m u m s u s t a i n e d p e r i o d f o r 64 FIGURE 4 O u t l i n e o f E x p e r i m e n t a l P r o c e d u r e . A. Main e x p e r i m e n t ( r e p r e s e n t s one o f two r e p l i c a t e s ) . Day 0 M a l e s s t r i p p e d , i n j e c t e d and i s o l a t e d , * = p o p u l a t i o n i n j e c t e d w i t h i s o t o p e . Day 5 M a l e s s t r i p p e d , i n j e c t e d and i s o l a t e d . Day 10 F e m a l e s ' o v i d u c t s f l u s h e d , a n a l r a y s c l i p p e d f o r i d e n t i f i c a t i o n and i s o l a t e d i n e x p e r i m e n t a l c a g e s . Day 25 M a l e s p l a c e d i n e x p e r i m e n t a l cage w i t h f e m a l e s . Day 32 E x p e r i m e n t t e r m i n a t e d , f e m a l e s ' o v i d u c t s s ampled f o r sperm, smears p r e p a r e d . B. L a b e l l e v e l c o n t r o l m a l e s . Day 0 M a l e s s t r i p p e d , i n j e c t e d and i s o l a t e d . Day 5~ M a l e s s t r i p p e d , i n j e c t e d and i s o l a t e d . Day 28 M a l e s s a m p l e d f o r sperm, smears p r e p a r e d , Day 32 M a l e s f r o m main e x p e r i m e n t sampled f o r sperm, smears p r e p a r e d . C. B a c k g r o u n d r a d i a t i o n c o n t r o l m a l e s . Day 0 M a l e s s t r i p p e d , i n j e c t e d w i t h s a l i n e s o l u t i o n and i s o l a t e d . Day 28 M a l e s s a m p l e d f o r sperm, smears p r e p a r e d . NOTE: T h i s r e p r e s e n t s one r e p l i c a t e o f two i n e a c h e x p e r i m e n t . I n t h e s e c o n d r e p l i c a t e t h e i s o t o p e i n j e c t e d p o p u l a t i o n o f males i s r e v e r s e d as a r e t h e l a b e l l e v e l c o n t r o l and b a c k g r o u n d r a d i a t i o n c o n t r o l m a l e s . The a n a l r a y s c l i p p e d i n f e m a l e s o f t h e two p o p u l a t i o n s a r e a l s o r e v e r s e d . 65-A B . C DAY 0 5 REPEAT REPEAT r o ~ i r~o~] 66 which high l e v e l s of l a b e l l e d sperm could be expected to be present i n injected males after the f i r s t of two injections was from day 22 to 32 post i n j e c t i o n . This i s due to the production of new and disintegration of old sperm i n the testes (Crowley, 1968) . On the other hand, L i l e y (1966) and my p i l o t results suggested that sperm i s accessible i n the oviduct for a period of approximately 7 days after which i t i s incorporated into the female's ovarian f o l d s . On the basis of these factors, the mating period was set at 7 days, from day 25 to day 32 of the experiment. VI. Assessment Procedures and Sources of Error Assessment Procedures Smears were examined under a l i g h t microscope with a micrometer g r i d using l i g h t - f i e l d i llumination. A smear was i n i t i a l l y examined for sperm at a magnification of 400X. Upon id e n t i f y i n g sperm, magnification was shifted to 1000X and the smear scanned u n t i l individual sperm could be i d e n t i f i e d . In some areas the density of sperm was too heavy to allow t h i s i d e n t i f i c a t i o n (see Figure 5). A sperm was considered l a b e l l e d i f one or more s i l v e r grains i n the emulsion overlying i t was activated ( i e . black grain). Beginning at the l e f t side of the micrometer g r i d , l a b e l l e d and unlabelled sperm were t a l l i e d using hand counters u n t i l a t o t a l of 100 sperm had been assessed, moving the g r i d to the right as many times as necessary. The smear was then again scanned u n t i l another area 67 i n w h i c h i n d i v i d u a l s p e r m c o u l d b e i d e n t i f i e d w a s f o u n d . A n o t h e r 100 s p e r m w e r e a s s e s s e d , l a b e l l e d a n d u n l a b e l l e d , a s d e s c r i b e d a b o v e . T h e s m e a r w a s t h e n s c a n n e d a t h i r d t i m e , a n d a f i n a l 100 s p e r m a s s e s s e d f o r l a b e l . T a l l i e s f o r e a c h s m e a r i n w h i c h s p e r m w a s p r e s e n t w e r e r e c o r d e d a s n u m b e r o f l a b e l l e d a n d u n l a b e l l e d s p e r m p e r 300. T h e s a m e a s s e s s m e n t p r o c e d u r e w a s u s e d f o r o v i d u c t s m e a r s , l a b e l l e v e l c o n t r o l s m e a r s a n d b a c k g r o u n d r a d i a t i o n c o n t r o l s m e a r s . F i g u r e 6 i l l u s t r a t e s a s m e a r s e c t i o n w i t h a l o w p r o p o r t i o n o f l a b e l l e d t o u n l a b e l l e d s p e r m , F i g u r e 7 i l l u s t r a t e s a s m e a r s e c t i o n w i t h a h i g h r a t i o o f l a b e l l e d t o u n l a b e l l e d s p e r m . M e a s u r e s o f M a t i n g S u c c e s s T w o c r i t e r i a w e r e e s t a b l i s h e d i n o r d e r t o d e t e r m i n e t h e m a t i n g s u c c e s s o f m a l e s p l a c e d i n c o m p e t i t i o n f o r f e m a l e s i n t h e f o u r e x p e r i m e n t s . A . N u m b e r o f A t t r i b u t a b l e I n s e m i n a t i o n s . I n a n u m b e r o f c a s e s i t w a s p o s s i b l e t o a t t r i b u t e t h e l a b e l l e d o r u n l a b e l l e d s p e r m p r e s e n t i n a s m e a r t o a m a l e ( s ) o f o n l y o n e o f t h e t w o p o p u l a t i o n s . T h e c r i t e r i a f o r a t t r i b u t i n g a n i n s e m i n a t i o n ( s ) t o o n e o f t h e p o p u l a t i o n s w e r e a s f o l l o w s : i f a s m e a r c o n t a i n e d o v e r 245/300 l a b e l l e d s p e r m i t w a s a t t r i b u t e d t o a m a l e ( s ) f r o m t h e i s o t o p e i n j e c t e d p o p u l a t i o n . S i m i l a r l y , i f t h e s m e a r c o n t a i n e d o v e r 284/300 u n l a b e l l e d s p e r m i t w a s a t t r i b u t e d t o a m a l e ( s ) f r o m t h e s a l i n e i n j e c t e d p o p u l a t i o n . T h e s e c r i t e r i a r e f e r e d t o a b o v e a r e b a s e d o n c o r r e c t i o n s f o r b a c k g r o u n d r a d i a t i o n a n d s p e r m l a b e l l e v e l i n i s o t o p e 68 FIGURE 5 Photomicrograph of female oviduct smear i l l u s t r a t i n g an area of heavy sperm concentration not suitable for t a l l y i n g . 1000X magnification. 69 70 FIGURE 6 Photomicrograph of female oviduct smear i l l u s t r a t i n g a low l e v e l of l a b e l l e d sperm. 1000X magnification. 7 1 72 FIGURE 7 Photomicrograph of female oviduct smear i l l u s t r a t i n g a high l e v e l of l a b e l l e d sperm. 1000X m a g n i f i c a t i o n . 73 74 injected males. Smears from males injected with saline i n the four experiments (background radiation controls) should contain no l a b e l l e d sperm. If activated grains over sperm are found i n these smears, the activation can be attributed to either background radiation or l i g h t contamination. Table 7 l i s t s mean number of sperm with activated s i l v e r halide grains over them t a l l i e d for the background radiation control smears for each re p l i c a t e of the four experiments. A grand mean has also been calculated. The t o t a l percent of sperm t a l l i e d as 'labelled* which may be due to background radiation i s 5.45%. The mean number of la b e l l e d sperm i n isotope injected males was also calculated for each re p l i c a t e . These means for males l a b e l l e v e l control smears are l i s t e d i n Table 8. The percentage of l a b e l l e d sperm for males over the four experiments, corrected for background radiation was 76.20%. This translates into 245/300 l a b e l l e d sperm, the lower l i m i t for an oviduct smear, and consequently an insemination, to be attributed to the isotope injected male(s). Conversely, oviduct samples contributed by the saline injected males should e x i b i t more than 284 unlabelled sperm. Up to 16 sperm associated with granules i n the emulsion (5.45%) can be attributed to background radiation. The number of attributable inseminations by males i n each re p l i c a t e were then combined and compared using the X2 Test (<< = 0.05, Snedecor and Cochran, 1967). B. Mean Number of Sperm. The measure of mating success discussed above does not incorporate the data from oviduct smears which appear to be the TABLE 7 Proportion of sperm appearing as 'labelled' in background radiation control smears. Experiment 1 2 3 4 Male population N G LA N LA G N P injected with saline Number of males 10 10 10 10 10 10 10 10 Total sperm t a l l i e d 300 300 300 300 300 300 300 300 per smear Mean 'label' due to 17.7 20.1 16.2 16.8 16.2 14.7 13.5 15.6 background radiation Mean 'label' due to 16.36 background radiation, a l l experiments % 'label' due to 5.45 background radiation, a l l experiments cn TABLE 8 P r o p o r t i o n of l a b e l l e d sperm i n i s o t o p e i n j e c t e d males, Experiment 1 2 3 4 Male p o p u l a t i o n N G LA N LA G N P i n j e c t e d w i t h s a l i n e Number of males 10 10 10 10 10 10 10 10 T o t a l sperm t a l l i e d 300 300 300 300 300 300 300 300 ^ per smear ^ Mean number of 238.2 231.2 229.1 230.6 261.7 255.6 254.4 258.9 l a b e l l e d sperm Mean number l a b e l l e d sperm f o r a l l e x periments 228.60 c o r r e c t e d f o r background r a d i a t i o n % o f l a b e l l e d sperm f o r a l l e xperiments c o r r e c t e d f o r 76.20 background r a d i a t i o n 77 r e s u l t of m u l t i p l e inseminations, that i s , those smears which co n t a i n l e s s than 245 l a b e l l e d sperm and those which contain l e s s than 284 u n l a b e l l e d sperm. These types of oviduct smears represent 64.4% of the oviduct smears found to contain sperm. In order to inco r p o r a t e the data i n these mixed oviduct smears, the t o t a l number of sperm c o n t r i b u t e d by males of the competing populations to females was compared using the Mann Whitney U t e s t (<* = 0.05, Snedecor and Cochran, 1967). The two sources of error discussed above could have a marked e f f e c t on the a c t u a l number of sperm c o n t r i b u t e d by males to females e x h i b i t i n g mixed samples i n the experiments. Background r a d i a t i o n could cause an overestimation of sperm c o n t r i b u t e d by isotope i n j e c t e d males and an underestimation of sperm c o n t r i b u t e d by s a l i n e i n j e c t e d males. L a b e l l e d sperm l e v e l s i n isotope i n j e c t e d males below 100% could conversely r e s u l t i n underestimating the isotope i n j e c t e d males' c o n t r i b u t i o n and overestimate the s a l i n e i n j e c t e d males* c o n t r i b u t i o n . In an attempt to overcome these p o s s i b l e sources of e r r o r . I ran two r e p l i c a t e s of each experiment w i t h the i n j e c t e d p o pulation of males reversed. This should e f f e c t i v e l y cancel out the e f f e c t s of the two sources of e r r o r , unless the e r r o r s were s i g n i f i c a n t l y d i f f e r e n t between r e p l i c a t e s . I t h e r e f o r e t e s t e d for d i f f e r e n c e s i n background r a d i a t i o n and male l a b e l l e v e l between r e p l i c a t e s f o r each experiment using Mann Whitney U Tests (<* = 0.05, Snedecor and Cochran, 1967). None of the comparisons were 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 . I t was the r e f o r e decided to use the raw scores and combine the r e p l i c a t e s i n comparing the mean number of sperm 78 c o n t r i b u t e d by males competing f o r females i n the four experiments. V I I . Results and Discussion Naranjo males were found to have inseminated more N females (p < 0.01) and c o n t r i b u t e d more sperm to N females (p < 0.001) than d i d Guayamare males (Figure 8A). Naranjo males were a l s o found to have inseminated more N females (p < 0.05) and co n t r i b u t e d more sperm to N females (p < 0.01) than Lower Aripo males ( f i g u r e 9B) . These r e s u l t s are c o n s i s t e n t w i t h the p r e d i c t i o n that headstream males u t i l i z i n g the d i s p l a y strategy i n t h e i r c o u r t s h i p would outcompete males u t i l i z i n g the t h r u s t strategy i n c l e a r water i n competition f o r headstream females. A s i m i l a r p r e d i c t i o n f o r midstream and lowland females was not supported, however. No s i g n i f i c a n t d i f f e r e n c e s were found i n the number of inseminations or the amount of sperm co n t r i b u t e d to G females by N and G males (Figure 8B). Neither were there any s i g n i f i c a n t d i f f e r e n c e s i n the number of inseminations and sperm c o n t r i b u t e d by N and LA males to LA females (Figure 9A) . These r e s u l t s suggest that w h i l e the d i s p l a y strategy (high d i s p l a y / l o w thrust) r e s u l t s i n greater mating success f o r headstream males with t h e i r own females, the t h r u s t strategy (low d i s p l a y / h i g h thrust) employed by midstream/lowland males i s equa l l y s u c c e s s f u l i n ensuring mating with midstream/lowland females. These r e s u l t s s t r o n g l y suggest that other f a c t o r s as w e l l as male d i s p l a y strategy may be involved i n determining mating success w i t h midstream and 79 lowland females. These w i l l , be discussed below. No s i g n i f i c a n t differences were found i n the number of inseminations and sperm contributed to females by males of the midstream LA and lowland G r i v e r s . This r e s u l t applies to both LA (Figure 10A) and G (Figure 10B) females. This supports my second prediction, that males from populations e x i b i t i n g similar courtship strategies from r i v e r s with similar physical conditions would not d i f f e r s i g n i f i c a n t l y i n the i r mating success with females of either population. Males from similar headstream rivers with similar courtship strategies did, however, mate asso r t a t i v e l y . Naranjo females where inseminated s i g n i f i c a n t l y more (p < 0.005) and had s i g n i f i c a n t l y more sperm contributed to them (p < 0.001) by Naranjo males than Paria males (Figure 11A). Conversely, P females exhibited the opposite trend, being inseminated more frequently (p < 0.05) and carrying more sperm (p < 0.001) from P males than N males (Figure 11B). The results from the four experiments suggest that headstream populations are capable of some degree of interpopulation discrimination, exercised by the female, male, or both. That t h i s discrimination i s based only on the female i n terms of preference for male courtship behaviour i s unlikely since the only s i g n i f i c a n t difference i d e n t i f i e d between the courtship behaviours of N and P males was the number of gonopodial swings, which are thought to be a displacement a c t i v i t i e s (Baerends, Brouwer and Waterbolk, 1955) or possibly intention movements (Crow, 1981). It i s also possible that N and P females are capable of distinguishing males of their own 80 population on the b a s i s of c o l o u r . Farr (1980) concluded from experiments w i t h inbred stocks of guppies t h a t male c o l o u r a t i o n could have some e f f e c t on male reproductive success, but only i n the absence of behavioural d i f f e r e n c e s . Haskins et a l . , (1961) a l s o found females to be capable of d i s c r i m i n a t i n g between males of d i f f e r e n t colour patterns but they d i d not c o n t r o l for behaviour. A t h i r d p o s s i b i l i t y e x i s t s , and that i s t h a t males of the two headstream populations are capable of d i s c r i m i n a t i n g f o r females of t h e i r own populations. B a l l i n (1973) suggested and Crow (1981) d i r e c t l y observed s i g n i f i c a n t preferences i n N males (Upper Aripo i n Crow) f o r N females and P males f o r P females i n experiments where the populations were observed together. These preferences were expressed as a higher number of d i s p l a y s and t h r u s t s d i r e c t e d by males at females of t h e i r own population. The mechanism for t h i s d i s c r i m i n a t i o n by males may be a population s p e c i f i c pheromone released by the female (Crow and L i l e y , 1979) . A high degree of i n t e r p o p u l a t i o n d i s c r i m i n a t i o n does not seem evident between the LA or G populations on the b a s i s of the r e s u l t s i l l u s t r a t e d i n Figure 10. Colour d i f f e r e n c e s between males of the LA and G populations are not as d i s t i n c t as those between the N and P and consequently females of the G and LA populations may be l e s s able to d i s t i n g u i s h males of ( t h e i r own pop u l a t i o n s . Crow (1981) also found t h a t w h i l e N and P males e x h i b i t e d a s i g n i f i c a n t preference i n t h e i r c o u r t s h i p toward t h e i r own females, G males were l e s s d i s c r i m i n a t i n g . As mentioned e a r l i e r , no s i g n i f i c a n t d i f f e r e n c e s were TABLE 9 Summary of Results of Mating Success in Clear Water Competition, Experiments 1 - 4 . Experiment Female Population Sampled Number of Females Recovered Number of Female Samples With Sperm Male Population Number of Samples Attributed to Male Population 60 28 N G 11 2 G 59 26 N G 6 6 LA 60 29 60 25 3 4 1 6 LA 59 21 58 27 LA N LA N LA G LA G 60 32 60 28 P N P 3 5 4 6 9 0 2 9 oo 82 FIGURE 8 Results of Experiment 1 A. Mean number of sperm per 300 and number of oviduct smears attributed to males of one population (crosshatched) contributed by Naranjo (N) and Guayamare (G) males to Naranjo females i n clear water. B. Mean number of sperm per 300 and number of oviduct smears attributed to males of one population (crosshatched) contributed by Naranjo (N) and Guayamare (G) males to Guayamare females i n clear water. a. Comparison of numbers of sperm; Mann Whitney U Test. b. Comparison of attributed samples, x2 Test. N.S. = not s i g n i f i c a n t at 0.05 l e v e l of significance 83 NUMBER OF ATTRIBUTED SAMPLES NARANJO FEMALES NUMBER OF ATTRIBUTED SAMPLES N.S. b GUAYAMARE FEMALES 84-FIGURE 9 R e s u l t s o f E x p e r i m e n t 2 A. Mean number of sperm p e r 300 and number of o v i d u c t smears a t t r i b u t e d t o m a l e s o f one p o p u l a t i o n ( c r o s s h a t c h e d ) c o n t r i b u t e d by Lower A r i p o (LA) and N a r a n j o (N) males t o Lower A r i p o f e m a l e s i n c l e a r w a t e r . B. Mean number o f sperm p e r 30 0 and number o f o v i d u c t smears a t t r i b u t e d t o m a l e s o f one p o p u l a t i o n ( c r o s s h a t c h e d ) c o n t r i b u t e d by Lower A r i p o (LA) and N a r a n j o (N) males t o N a r a n j o f e m a l e s i n c l e a r w a t e r . a. C o m p a r i s o n o f numbers o f sperm; Mann W h i t n e y U T e s t . b. C o m p a r i s o n o f a t t r i b u t e d s a m p l e s , x2 T e s t . N.S. = n o t s i g n i f i c a n t a t 0.05 l e v e l o f s i g n i f i c a n c e 85 300—| 200-—^ MEAN NUMBER SPERM PER 300 100-H N.S. N.S. LOWER ARIPO FEMALES NUMBER OF ATTRIBUTED SAMPLES 300—i 200—4 MEAN NUMBER SPERM PER 300 p < 0.01c p < 0.05 u NARANJO FEMALES F—20 •30 NUMBER OF ATTRIBUTED SAMPLES -10 86 FIGURE 10 Results of Experiment 3 A. Mean number of sperm per 300 and number of oviduct smears attributed to males of one population (crosshatched) contributed by Lower Aripo (LA) and Guayamare (G) males to Lower Aripo females i n clear water. B. Mean number of sperm per 300 and number of oviduct smears attributed to males of one population (crosshatched) contributed by Lower Aripo (LA) and Guayamare (G) males to Guayamare females i n clear water. a. Comparison of numbers of sperm; Mann Whitney U Test. b. Comparison of attributed samples, x2 Test. N.S. = not s i g n i f i c a n t at 0.05 l e v e l of significance 87 300 200—^ MEAN NUMBER SPERM PER 300 N.S. N.S. LOWER ARIPO FEMALES NUMBER OF ATTRIBUTED SAMPLES 300—, 200-MEAN NUMBER SPERM PER 300 100-H N.S. NUMBER OF ATTRIBUTED SAMPLES N.S. GUAYAMARE FEMALES 88 FIGURE 11 Results of Experiment 4 A. Mean number of sperm per 300 and number of oviduct smears attributed to males of one population (crosshatched) contributed by Naranjo (N) and Paria (P) males to Naranjo females i n clear water. B. Mean number of sperm per 300 and number of oviduct smears attributed to males of one population (crosshatched) contributed by Naranjo (N) and Paria (P) males to Paria females i n clear water. a. Comparison of numbers of sperm; Mann Whitney U Test. b. Comparison of attributed samples, x2 Test. N.S. = not s i g n i f i c a n t at 0.05 l e v e l of significance 89 300 — I 30 p < 0.001c 200.-3 MEAN NUMBER SPERM PER 300 100-p < 0.005 NARANJO FEMALES •20 NUMBER OF ATTRIBUTED SAMPLES 10 300-p < O.OOl' r—30 200-MEAN NUMBER SPERM PER 300 100-p < 0 . 0 5 ° PARIA FEMALES (—20 NUMBER OF ATTRIBUTED SAMPLES -10 90 f o u n d b e t w e e n t h e m a t i n g s u c c e s s o f N m a l e s a n d G m a l e s w i t h G f e m a l e s n o r w e r e a n y f o u n d b e t w e e n t h e m a t i n g s u c c e s s o f N m a l e s a n d L A m a l e s w i t h L A f e m a l e s . T h e s e r e s u l t s m a y b e d u e t o t h e r e l a t i v e l y h i g h l e v e l s o f c o h e s i v e n e s s o b s e r v e d i n t h e G a n d L A p o p u l a t i o n s . G a n d L A f e m a l e s c o u l d b e e x p e c t e d t o e n c o u n t e r m a l e s o f t h e i r o w n p o p u l a t i o n m o r e f r e q u e n t l y t h a n t h o s e o f t h e N p o p u l a t i o n . T h i s m a y h a v e c o u n t e r b a l a n c e d a n y f e m a l e p r e f e r e n c e f o r h i g h e r d i s p l a y i n g m a l e s . V I I I . S u m m a r y o f R e s u l t s M a l e s o f f o u r p o p u l a t i o n s o f P o e c i l i a r e t i c u l a t a , t w o f r o m c l e a r h e a d s t r e a m r i v e r s ( N a r a n j o a n d P a r i a ) , o n e f r o m a t u r b i d l o w l a n d r i v e r ( G u a y a m a r e ) a n d a n o t h e r f r o m a n i n t e r m e d i a t e m i d s t r e a m r i v e r ( L o w e r A r i p o ) w e r e p l a c e d i n c o m p e t i t i o n f o r m a t i n g w i t h f e m a l e s o f t h e s e p o p u l a t i o n s . F o u r e x p e r i m e n t s w e r e d e s i g n e d t o t e s t t h e h y p o t h e s i s t h a t d i f f e r e n c e s i n c o h e s i o n a n d m a t i n g s t r a t e g i e s b e t w e e n t h e s e p o p u l a t i o n s a r e a d a p t i o n s , i n p a r t , t o d i f f e r e n c e s i n t h e t u r b i d i t y o f t h e r i v e r s i n w h i c h t h e s e p o p u l a t i o n s n a t u r a l l y o c c u r . 1. T h e p r e d i c t i o n t h a t m a l e s f r o m c l e a r h e a d s t r e a m p o p u l a t i o n s ( N a n d P ) w o u l d b e m o r e s u c c e s s f u l a t m a t i n g t h a n m a l e s f r o m t h e t u r b i d m i d s t r e a m a n d l o w l a n d p o p u l a t i o n s ( G a n d L A ) w h e n p l a c e d i n c o m p e t i t i o n f o r h e a d s t r e a m f e m a l e s i n c l e a r w a t e r w a s s u p p o r t e d . 2. T h e p r e d i c t i o n t h a t m a l e s f r o m h e a d s t r e a m p o p u l a t i o n s w o u l d b e m o r e s u c c e s s f u l a t m a t i n g t h a n m a l e s f r o m m i d s t r e a m a n d l o w l a n d p o p u l a t i o n s w h e n p l a c e d i n c o m p e t i t i o n f o r m i d s t r e a m 91 and lowland females i n clear water was not supported. It was suggested that greater cohesion i n midstream and lowland populations could have counteracted a preference of females of these populations for higher display frequency and duration exhibited by headstream males. 3. The prediction that there would be no s i g n i f i c a n t differences i n mating success between males of turbid midstream (LA) and turbid lowland (G) r i v e r s when placed i n competition for females of both populations i n clear water was supported. 4. The prediction that there would be no s i g n i f i c a n t differences between the mating success of two clear water headstream populations (N and P) when placed i n competition for females of both populations i n clear water was not supported. The results indicated interpopulation discrimination by both populations. It was suggested that this discrimination could be exercised on the basis of female preference for colour, or male preference on the basis of a pheromone or both. The results suggest that increased display frequency and duration i n headstream males are, in part, adaptions to the clear water conditions evident i n their natural environment. 92 C H A P T E R F I V E M A T I N G S U C C E S S O F M A L E S I N C O M P E T I T I O N F O R F E M A L E S I N T U R B I D W A T E R I . I n t r o d u c t i o n R e s u l t s i n C h a p t e r 5 s h o w t h a t h e a d s t r e a m m a l e s e x h i b i t i n g h i g h f r e q u e n c i e s a n d d u r a t i o n s o f d i s p l a y i n t h e i r c o u r t s h i p a r e m o r e s u c c e s s f u l a t i n s e m i n a t i n g f e m a l e s o f t h e i r o w n p o p u l a t i o n s t h a n a r e m a l e s e x i b i t i n g l o w l e v e l s o f d i s p l a y a n d h i g h f r e q u e n c i e s o f t h r u s t s , i n c l e a r w a t e r . H e a d s t r e a m m a l e s e m p l o y i n g t h e d i s p l a y s t r a t e g y a r e a l s o e q u a l l y s u c c e s s f u l a t i n s e m i n a t i n g f e m a l e s f r o m m i d s t r e a m a n d l o w l a n d p o p u l a t i o n s a s a r e t h r u s t s t r a t e g y m a l e s f r o m t h o s e p o p u l a t i o n s , i n c l e a r w a t e r . T h e s e r e s u l t s a r g u e s t r o n g l y i n f a v o u r o f t h e h y p o t h e s i s t h a t h i g h f r e q u e n c y a n d d u r a t i o n o f d i s p l a y i n m a l e c o u r t s h i p a r e a d a p t a t i o n s t o t h e c l e a r w a t e r c o n d i t i o n s i n w h i c h t h e s e m a l e s e x i s t . E p i g a m i c s e x u a l s e l e c t i o n i s s e e n a s a s t r o n g s e l e c t i v e f a c t o r i n t h e s e c l e a r w a t e r r i v e r s . T u r b i d i t y a n d p r e d a t i o n a r e b o t h p r e s s u r e s e x i s t i n g i n l o w l a n d r i v e r s w h i c h p r e s u m a b l y c o u n t e r a c t s e x u a l s e l e c t i o n r e s u l t i n g i n b r i g h t c o l o u r , l a r g e s i z e a n d h i g h f r e q u e n c y a n d d u r a t i o n o f d i s p l a y . O n e w o u l d t h e r e f o r e e x p e c t f i s h i n l o w l a n d p o p u l a t i o n s t o b e s m a l l e r , l e s s b r i g h t l y c o l o u r e d , m o r e c o h e s i v e a n d e x i b i t c o u r t s h i p b e h a v i o u r r e l y i n g l e s s o n t h e v i s u a l c o m p o n e n t , t h a t i s , t h e d i s p l a y s t r a t e g y . O b s e r v a t i o n s o f b e h a v i o u r i n l o w l a n d a n d m i d s t r e a m p o p u l a t i o n s i n t h e f i e l d 93 and the l a b o r a t o r y support these p r e d i c t i o n s (see Chapter 3 ) . The experiment described i n t h i s chapter was designed to t e s t . the hypothesis t h a t cohesiveness and a high frequency of t h r u s t s i n male c o u r t s h i p behaviour are adaptions, i n p a r t , to the t u r b i d water c o n d i t i o n s experienced by f i s h i n lowland r i v e r s . T u r b i d i t y , as determined by the two measures described i n Chapter 2, was found to be s i g n i f i c a n t l y higher i n the lowland Guayamare River than i n the headstream Naranjo River (Mann Whitney U Test, Snedecor and Cochran, 1967; Sechi, p < 0.01; meter, p < 0.01). On the b a s i s of the hypothesis I p r e d i c t e d t h a t : 1. Males from the t u r b i d lowland Guayamare r i v e r would be more s u c c e s s f u l at mating than males from the c l e a r headstream Naranjo r i v e r when placed i n competition f o r females of both populations i n t u r b i d water c o n d i t i o n s . This p r e d i c t i o n was t e s t e d i n Experiment 5, using the sperm l a b e l l i n g and assessment methods described i n Chapter 4. I I . Experimental Design and Procedure I c o l l e c t e d guppies from f i v e r i v e r s i n T r i n i d a d , West Ind i e s , i n c l u d i n g the Naranjo and Guayamare, and shipped these to the U n i v e r s i t y of B r i t i s h Columbia, Vancouver. The stocks were housed, by L i l e y and Crow, i n separate tanks i n three c o n t r o l l e d l a b o r a t o r i e s . The tanks i n which they were held were of 43 and 61 1 capacity with g r a v e l substrates and planted with V a l l i s n e r i a and Lemna. A l l tanks were maintained under 30. or 40 watt f l u o r e s c e n t tubes and f i l t e r e d 94 by s u b g r a v e l , and i n t e r n a l and e x t e r n a l c h a r c o a l f i l t e r s . One t h i r d o f t h e w a t e r i n e a c h t a n k was r e g u l a r l y c h anged w i t h d e c h l o r i n a t e d t a p w a t e r . A l l t h r e e l a b o r a t o r i e s were on a 13 h l i g h t - 11 h d a r k p h o t o p e r i o d and m a i n a t a i n e d a t 23 - 26 °C. Young were r e g u l a r l y removed f r o m t h e w i l d c a u g h t t a n k s . A l l f i s h were f e d on a c o m m e r c i a l l y p r e p a r e d d r i e d f i s h f o o d , T e t r a - m i n , o c c a s s i o n a l l y s u p p l e m e n t e d w i t h b r i n e s h r i m p or T u b i f e x worms. T h i s e x p e r i m e n t was c a r r i e d o u t a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a i n two t a n k s d e s i g n e d by L i l e y and Crow ( F i g u r e 1 2 ) . The t a n k s were c o n s t r u c t e d o u t o f p l e x i g l a s s and had a p l y w o o d b a s e . E a c h tank h e l d a p p r o x i m a t e l y 500 1 o f w a t e r and was a e r a t e d u s i n g a i r s t o n e s . Water f l o w c o u l d be c o n t r o l l e d t h r o u g h t h e use o f a s u b m e r s i b l e pump (Model 4SMD, L i t t l e G i a n t Pump Company) w h i c h was r e g u l a t e d by a v a r i a b l e t r a n s f o r m e r ( P o w e r s t a t , Type 3PN, S u p e r i o r E l e c t r i c a l Company). Crow (1981) p r o v i d e s a d e t a i l e d d e s c r i p t i o n o f t h i s c o n t r o l s y s t e m . S e v e r a l a l t e r n a t i v e s were t r i e d i n a t t e m p t i n g t o make t h e w a t e r i n t h e t a n k s t u r b i d , t h e most s u c c e s s f u l b e i n g t h e p a r t i c u l a t e i n w a t e r c o l l e c t e d f r o m s t r e a m s a l o n g t h e C a p i l a n o M o u n t a i n Road, N o r t h V a n c o u v e r . S u b s t r a t e f r o m t h e s e s t r e a m s was c o l l e c t e d i n p l a s t i c b u c k e t s and d e c h l o r i n a t e d t a p w a t e r added. The s u b s t r a t e was t h e n s t i r r e d and a l l o w e d t o s e t t l e f o r 1 h o u r . The w a t e r w i t h t h e s u s p e n d e d p a r t i c u l a t e was t h e n added t o t h e t a n k w a t e r . A s l i g h t c u r r e n t e s t a b l i s h e d i n e a c h t a n k h e l p e d t o keep t h e p a r t i c u l a t e s u s p e n d e d . A w h i t e d i s k s i m i l a r t o t h e one u s e d i n m e a s u r i n g 95 FIGURE 12 E x p e r i m e n t a l tank used i n Experiment 5. 1. S u b m e r s i b l e pump. 2. P l a s t i c t ube. 3. P l e x i g l a s s tube w i t h 6 mm h o l e s . 4. V a r i a b l e a u t o - t r a n s f o r m e r . Adapted from Crow, 1981. 96 WO OOI 97 t u r b i d i t y i n T r i n i d a d streams and a p l a s t i c metric r u l e r were used to measure t u r b i d i t y . T u r b i d i t y was checked twice a day during the experimental period and maintained at 6± 2 cm. This was done by removing water from the experimental tanks and adding t u r b i d d e c h l o r i n a t e d water. Water temperature was mainatined at 22 - 24 °C. The lab o r a t o r y was on a 13 h l i g h t - 11 h dark photoperiod and each tank was l i t by 8 40 watt f l u o r e s c e n t tubes suspended 2 m above the tank. The autoradiographic methods used i n t h i s experiment was. the same as those used i n the four c l e a r water experiments conducted i n T r i n i d a d (Chapter 3, 'sections I I - IV ) . One competitive mating experiment, designated '5', was conducted i n t u r b i d water. Wild caught males from the Naranjo and Guayamare populations were placed i n competition f o r mating with females of these same populations. D e t a i l s of the design are l i s t e d i n Table 10. Two r e p l i c a t e s were run i n the experiment, r e v e r s i n g the isotope i n j e c t e d population of males and the i d e n t i f i c a t i o n mark on females. These r e p l i c a t e s were run simultaneously i n two separate experimental tanks and the data l a t e r combined f o r a n a l y s i s . On day 0 of the experiment 25 Naranjo males were removed from the stock tank, were s t r i p p e d and i n j e c t e d with 2.0 u l of thymidine - (methyl-3H)- at a concentration of 1.0 cu/1. 30 Guayamare males were s t r i p p e d and i n j e c t e d with 2.0 u l s a l i n e (0.6% NaCI) s o l u t i o n . These males were then i s o l a t e d i n separate holding tanks. A f u r t h e r 30 Naranjo males and Guayamare males were removed from the stock tanks and processed 98 TABLE 10. Mating Success in Turbid Water Competition, Experimental Outline (5) EXPERIMENT POPULATION TYPE Headstream Lowland River COMPETING POPULATIONS Naranjo Guayamare REPLICATE FEMALE POPULATION FEMALES MATED 30 30 30 30 PEDUNCLE MARK top bottom bottom top MALE POPULATION MALES MATED 20 20 20 20 EXPERIMENTAL TANK a indicates male population injected with isotope. 99 as above, except with the i n j e c t i o n s reversed, f o r r e p l i c a t e 2. On day 5 of the experiment the s t r i p p i n g and i n j e c t i o n procedure was repeated for a l l males. On day 10 a t h i r d i n j e c t i o n was given to a l l males i n an attempt to lengthen the per i o d over which high l e v e l s of l a b e l l e d sperm would be present. On day 10, 35 females from each of the populations were removed from the stock tanks and marked f o r l a t e r i d e n t i f i c a t i o n . The marking procedure i n v o l v e d i n j e c t i n g a small amount of Trypan blue i n t o the musculature of the caudal peduncle of the female. Naranjo females were marked with a dot at the top of the peduncle, Guayamare females w i t h a dot at the bottom. A f u r t h e r 35 females of each population were removed from the stock tanks and marked (reversed) for r e p l i c a t e 2. The oviducts of a l l females were f l u s h e d using a micro p i p e t t e and s a l i n e s o l u t i o n . The females were then i s o l a t e d i n separate tanks and these tanks checked r e g u l a r l y for young, which i f found, were removed. On day 22 t u r b i d water was added to the experimental tanks and t u r b i t y brought to 6 cm. On day 25, 30 of the females and 20 males of each population designated for each of the r e p l i c a t e s were placed i n the r e s p e c t i v e experimental tank (Table 10). The ten remaining s a l i n e i n j e c t e d males f o r each r e p l i c a t e and the f i v e remaining isotope i n j e c t e d males f o r each r e p l i c a t e were s t r i p p e d on day 28 i n order to prepare background r a d i a t i o n c o n t r o l smears and l a b e l l e v e l c o n t r o l smears r e s p e c t i v e l y (see Chapter 5, s e c t i o n V) . 100 O n d a y 32 t h e c o m p e t i t i v e m a t i n g e x p e r i m e n t w a s t e r m i n a t e d , a l l f i s h m e a s u r e d , o v i d u c t s m e a r s p r e p a r e d f r o m f e m a l e s , a n d a f u r t h e r f i v e i s o t o p e i n j e c t e d m a l e s s t r i p p e d i n o r d e r t o p r e p a r e l a b e l l e v e l c o n t r o l s m e a r s . A l l s m e a r s w e r e t h e n p r o c e s s e d , a s s e s s e d a s d e s c r i b e d i n C h a p t e r 5, s e c t i o n V I , a n d t h e f i s h k i l l e d i n a h i g h c o n c e n t r a t i o n o f MS 222. T h e e s s e n t i a l e l e m e n t s o f t h e e x p e r i m e n t a l p r o c e d u r e a r e d e s c r i b e d i n C h a p t e r 3 a n d i l l u s t r a t e d i n F i g u r e 4 w i t h t h e e x c e p t i o n s d i s c u s s e d a b o v e . A s s e s s m e n t p r o c e d u r e s u s e d f o r d e t e r m i n i n g m a t i n g s u c c e s s i n e x p e r i m e n t 5 w e r e t h e s a m e a s t h o s e f o r e x p e r i m e n t s 1 - 4 d e s c r i b e d i n C h a p t e r 3, s e c t i o n V I . T h e p e r c e n t a g e o f s p e r m c o u n t e d a s l a b e l l e d a t t r i b u t a b l e t o b a c k g r o u n d r a d i a t i o n w a s 5.47% a n d t h e l e v e l o f l a b e l l e d s p e r m i n i s o t o p e i n j e c t e d m a l e s , c o r r e c t e d f o r b a c k g r o u n d r a d i a t i o n w a s 73.9%. T h u s , i n e s t i m a t i n g i n s e m i n a t i o n s a t t r i b u t a b l e t o m a l e s o f c o m p e t i n g p o p u l a t i o n s , s m e a r s c o n t a i n i n g o v e r 23 8/300 l a b e l l e d s p e r m w e r e a t t r i b u t e d t o m a t i n g s i n v o l v i n g o n l y m a l e s o f t h e l a b e l l e d p o p u l a t i o n , w h i l e t h o s e e x i b i t i n g o v e r 283/300 u n l a b e l l e d s p e r m w e r e a t t r i b u t e d t o m a t i n g s i n v o l v i n g o n l y t h e u n l a b e l l e d p o p u l a t i o n . T h e n u m b e r o f a t t r i b u t a b l e i n s e m i n a t i o n s b y m a l e s o f e a c h p o p u l a t i o n i n t h e r e p l i c a t e s w a s t h e n c o m b i n e d a n d c o m p a r e d u s i n g t h e x2 T e s t (<x = 0.05, S n e d a c o r a n d C o c h r a n , 1967) . I n o r d e r t o i n c o r p o r a t e t h e d a t a f r o m o v i d u c t s m e a r s w h i c h i n d i c a t e d m u l t i p l e i n s e m i n a t i o n s (51.06% o f t h e o v i d u c t s m e a r s ) t h e t o t a l n u m b e r o f s p e r m c o n t r i b u t e d b y m a l e s o f t h e c o m p e t i n g p o p u l a t i o n s t o f e m a l e s w a s c o m p a r e d u s i n g t h e M a n n W h i t n e y U 101 Test ( c < = 0.05, Snedecor and Cochran, 1967). Since no s i g n i f i c a n t differences were found i n background radiation and male la b e l l e v e l between the repl i c a t e s (p > 0.05, Mann Whitney U Test, Snedecor and Cochran, 1967), i t was assumed that combining the repl i c a t e s would cancel any error i n t a l l y i n g due to these factors. It was therfore decided to run the tests on the combined raw t a l l i e s . I I I . Results and Discussion Guayamare males were s i g n i f i c a n t l y more successful at mating with Guayamare females i n turbid water i n terms of both inseminations attributable to one population (p < 0.005) and t o t a l number of sperm (p < 0.001) than were Naranjo males (Figure 13B). There was no s i g n i f i c a n t difference i n the number of attributable inseminations by G and N males with N females. Naranjo males did, however, contribute s i g n i f i c a n t l y more sperm to N females than did G males (p < 0.01, Figure 13A). These results confirm the prediction that G males would be more successful at mating with G females i n turbid water conditions. While N males were s t i l l found to be more successful with their own females i n terms of t o t a l number of sperm contributed, comparing results i n clear water (Figure 8A) and those for turbid water (Figure 13A) suggest that G males have increased t h e i r mating success considerably i n turbid water. When comparing the number of samples attribuatable to one population of males between clear water (Table 9, experiment 1) and turbid (Table 11), the number i n N females 102 FIGURE 13 Results of Experiment 5 A. Mean number of sperm per 300 and number of oviduct smears attributed to males of one population (crosshatched) contributed by Naranjo (N) and Guayamare (G) males to Naranjo females i n turbid water. B. Mean number of sperm per 300 and number of oviduct smears attributed to males of one population (crosshatched) contributed by Naranjo (N) and Guayamare (G) males to Guayamare females i n turbid water. a. Comparison of numbers of sperm; Mann Whitney U Test. b. Comparison of attributed samples, x2 Test. N.S. = not s i g n i f i c a n t at 0.05 l e v e l of significance 103 300 200-MEAN NUMBER SPERM PER 300 100H p < 0.01 s' N . S . U NARANJO FEMALES •30 20 NUMBER OF ATTRIBUTED SAMPLES h-io 300-200-MEAN NUMBER SPERM PER 300 100-H p < 0.001c p < 0.005u GUAYAMARE FEMALES •30 -20 NUMBER OF ATTRIBUTED SAMPLES r-10 104 TABLE 11 Summary of Results of Mating Success in Turbid u Water Competition, Experiment 5. Experiment 5 F e S a m p l e d P U l a t i ° n NARANJO GUAYAMARE Number of Females Recovered 59 60 Number of Female Samples With 25 22 Sperm Male Population N G N G Number of Samples Attributed to 3 5 2 13 Male Population 105 a t t r i b u t a b l e t o N males has d r o p p e d f r o m 11 t o 3 w h i l e t h e number a t t r i b u t a b l e t o G males i n c r e a s e d f r o m 2 t o 5. T h i s a l s o s u g g e s t s t h a t G males a r e more s u c c e s s f u l i n t u r b i d w a t e r a t m a t i n g w i t h f e m a l e s f r o m b o t h p o p u l a t i o n s t h a n i n c l e a r w a t e r . T h i s may be due t o an i n c r e a s e d s u c c e s s i n i n s e m i n a t i o n by t h r u s t i n g w i t h b o t h N and G f e m a l e s . The g r e a t e r c o h e s i o n o f G f i s h w o u l d a l s o i n c r e a s e t h e s u c c e s s o f G m a l e s i n m a t i n g w i t h G f e m a l e s . The r e s u l t s s u p p o r t t h e h y p o t h e s i s t h a t a h i g h f r e q u e n c y o f g o n o p o d i a l t h r u s t s i n t h e c o u r t s h i p o f l o w l a n d males and g r e a t e r c o h e s i o n i n l o w l a n d p o p u l a t i o n s a r e , i n p a r t , a d a p t a t i o n s t o t h e t u r b i d w a t e r c o n d i t i o n s i n w h i c h t h e p o p u l a t i o n s e x i s t . IV. Summary o f R e s u l t s M a l e s o f two p o p u l a t i o n s o f P o e c i l i a r e t i c u l a t a , one f r o m a c l e a r h e a d s t r e a m r i v e r ( N a r a n j o ) and one f r o m a t u r b i d - l o w l a n d r i v e r (Guayamare) were p l a c e d i n c o m p e t i t i o n f o r m a t i n g w i t h f e m a l e s o f b o t h p o p u l a t i o n s . An e x p e r i m e n t was d e s i g n e d t o t e s t t h e h y p o t h e s i s t h a t d i f f e r e n c e s i n c o h e s i o n and m a t i n g s t r a t e g i e s between t h e s e p o p u l a t i o n s a r e a d a p t i o n s , i n p a r t , t o d i f f e r e n c e s i n t h e t u r b i d i t y o f t h e r i v e r s i n w h i c h t h e s e p o p u l a t i o n s n a t u a r a l l y o c c u r . 1. The p r e d i c t i o n t h a t m a l e s f r o m t h e t u r b i d l o w l a n d p o p u l a t i o n (G) would be more s u c c e s s f u l a t m a t i n g t h a n m a l e s f r o m t h e c l e a r h e a d s t r e a m p o p u l a t i o n (N) when p l a c e d i n c o m p e t i t i o n f o r l o w l a n d f e m a l e s i n t u r b i d w a t e r was s u p p o r t e d . 106 2., T h e p r e d i c t i o n t h a t m a l e s f r o m t h e l o w l a n d p o p u l a t i o n w o u l d b e m o r e s u c c e s s f u l a t m a t i n g t h a n m a l e s f r o m t h e h e a d s t r e a m p o p u l a t i o n w h e n p l a c e d i n c o m p e t i t i o n f o r h e a d s t r e a m f e m a l e s i n t u r b i d w a t e r w a s n o t s u p p o r t e d . H e a d s t r e a m m a l e s c o n t r i b u t e d s i g n i f i c a n t l y m o r e s p e r m t o h e a d s t r e a m f e m a l e s , a l t h o u g h n o t a s m u c h a s i n c l e a r w a t e r . A l s o , t h e n u m b e r o f a t t r i b u t a b l e m a t i n g s b y m a l e s o f o n e p o p u l a t i o n t o h e a d s t r e a m f e m a l e s w a s n o t s i g n i f i c a n t l y g r e a t e r f o r h e a d s t r e a m m a l e s a s c o m p a r e d t o l o w l a n d m a l e s . 3. I n c o m p a r i n g t h e m a t i n g s u c c e s s o f h e a d s t r e a m m a l e s a n d l o w l a n d m a l e s i n c l e a r a n d t u r b i d w a t e r , i t w a s c l e a r t h a t o v e r a l l m a t i n g s u c c e s s o f h e a d s t r e a m m a l e s d e c r e a s e d f r o m c l e a r t o t u r b i d w h i l e o v e r a l l m a t i n g s u c c e s s f o r l o w l a n d m a l e s i n c r e a s e d f r o m c l e a r t o t u r b i d w a t e r . 4. T h e s e r e s u l t s s u g g e s t t h a t h i g h d i s p l a y f r e q u e n c y a n d d u r a t i o n a r e a d a p t a t i o n s , i n p a r t , t o c l e a r w a t e r c o n d i t i o n s a n d t h a t c o h e s i o n a n d h i g h t h r u s t f r e q u e n c y a r e a d a p t a t i o n s t o t u r b i d w a t e r c o n d i t i o n s . 107 C H A P T E R S I X G E N E R A L D I S C U S S I O N T H E A D A P T I V E S I G N I F I C A N C E O F G E O G R A P H I C V A R I A T I O N I N T H E B E H A V I O U R A N D M O R P H O L O G Y O F P o e c i l i a r e t i c u l a t a S t u d i e s i n v e s t i g a t i n g t h e b e h a v i o u r a n d m o r p h o l o g y o f P o e c i l i a r e t i c u l a t a h a v e b e e n c o n c e r n e d t o a l a r g e d e g r e e w i t h c o r r e l a t i n g v a r i a t i o n i n t h e s e c h a r a c t e r i s t i c s w i t h d i f f e r e n c e s i n t h e b i o t i c a n d a b i o t i c c o n d i t i o n s i n w h i c h n a t u r a l p o p u l a t i o n s e x i s t . T h e a i m o f t h e s e s t u d i e s i s t o i d e n t i f y p o s s i b l e s e l e c t i v e a g e n t s i n t h e e n v i r o n m e n t s o f t h e g u p p y w h i c h m a y , i n a n e v o l u t i o n a r y s e n s e , b e r e s p o n s i b l e f o r t h e o b s e r v e d v a r i a t i o n . T h i s c h a p t e r s e r v e s f i r s t a s a s u m m a r y o f t h e r e s u l t s o f t h i s s t u d y . S e c o n d l y , i t i n c o r p o r a t e s t h e s e r e s u l t s i n t o t h e l a r g e r f r a m e w o r k o f r e s e a r c h i n v e s t i g a t i n g t h e a d a p t i v e s i g n i f i c a n c e o f b o t h b e h a v i o u r a l a n d m o r p h o l o g i c a l c h a r a c t e r i s t i c s i n t h i s s p e c i e s . F i n a l l y , i t o u t l i n e s p o t e n t i a l f u t u r e d i r e c t i o n s f o r r e s e a r c h a i m e d a t g a i n i n g a m o r e c o m p r e h e n s i v e u n d e r s t a n d i n g o f t h e ' f u n c t i o n s ' o f b e h a v i o u r i n P o e c i l i a r e t i c u l a t a . D i f f e r e n c e s w e r e f o u n d t o e x i s t i n t h e a b i o t i c c h a r a c t e r i s t i c s o f f o u r r i v e r s i n T r i n i d a d i n w h i c h g u p p i e s e x i s t , n a m e l y , t w o g e o g r a p h i c a l l y i s o l a t e d h e a d s t r e a m r i v e r s , t h e N a r a n j o a n d P a r i a ; a m i d s t r e a m r i v e r , t h e L o w e r A r i p o ; a n d a l o w l a n d r i v e r , t h e G u a y a m a r e . T u r b i d i t y i n p a r t i c u l a r w a s f o u n d t o i n c r e a s e m a r k e d l y f r o m t h e h e a d s t r e a m t o t h e l o w l a n d 108 r i v e r s . Male guppies can attempt to inseminate females through two courtship strategies, the f i r s t i s through display, the other through thrusting. The display strategy involves active female p a r t i c i p a t i o n , the thrust strategy does not. In laboratory observations, B a l l i n (1973) f i r s t i d e n t i f i e d differences i n the courtship behaviour of male guppies from clear headstream ri v e r s and males from turbid lowland r i v e r s . He found headstream males to exhibit displays at higher frequencies and for longer durations than lowland males while the l a t t e r exibited thrusts at higher frequencies than headstream males. B a l l i n speculated that the lower display rate and higher thrust rate observed i n lowland males r e l a t i v e to that observed i n headstream males was a response to heavy predation i n lowland r i v e r s . I suggested that these differences could also be responses to differences i n t u r b i d i t y between the r i v e r s , with males i n the clear headstreams relying more heavily on the vi s u a l display strategy in their courtship and males from turbid r i v e r s relying more on the les s v i s u a l l y oriented thrust strategy. Seghers (1973) observed i n laboratory observations that f i s h from lowland r i v e r s are more cohesive and inhabit areas closer to shore than those i n headstream r i v e r s . He interpreted these behaviours i n lowland f i s h as also being anti-predator adaptations. High cohesiveness can also be interpreted as a response to t u r b i d i t y , as i t would f a c i l i t a t e the location of mates during courtship under turbid water conditions. The f i r s t objective of t h i s study was to confirm that the 109 d i f f e r e n c e s i n behaviour i d e n t i f i e d by B a l l i n (1973) and Seghers (1973) are present i n f i s h i n the n a t u r a l environment. Guppies were observed i n the low predator, c l e a r Naranjo and P a r i a headstreams r i v e r s , the high predator, t u r b i d lowland Guayamare River and the intermediate Lower Aripo R i v e r . D i f f e r e n c e s i n the behaviour of populations of guppies observed i n the four r i v e r s were i n the p r e d i c t e d d i r e c t i o n . F i s h i n the headstream r i v e r s were l e s s cohesive and i n h a b i t e d \ areas f u r t h e r from shore than d i d lowland f i s h . In t h e i r c o u r t s h i p behaviour, headstream males performed sigmoid d i s p l a y s at a higher frequency and f o r longer durations than d i d midstream or lowland males. Conversely, the lowland males were found to e x h i b i t a higher frequency of gonopodial t h r u s t s than d i d males of the other three r i v e r s . I a l s o attempted to determine i f the observed d i f f e r e n c e s i n behaviour were, i n p a r t , due to genetic d i f f e r e n c e s . Guppies from the populations observed i n the f i e l d were bred and r a i s e d under i d e n t i c a l c o n d i t i o n s i n the l a b o r a t o r y and c o u r t s h i p behaviour observed i n males. I p r e d i c t e d that the d i f f e r e n c e s observed i n the f i e l d would p e r s i s t i n the l a b o r a t o r y r a i s e d f i s h . A l l of the behavioural d i f f e r e n c e s observed i n the f i e l d , save one, were found to p e r s i s t i n the l a b o r a t o r y r a i s e d f i s h . The d i f f e r e n c e i n the frequency of t h r u s t s e x h i b i t e d by Guayamare males (higher) as compared to Lower Aripo males observed i n the f i e l d was the only comparison not found to be s t a t i s t i c a l l y s i g n i f i c a n t i n the l a b o r a t o r y r a i s e d stocks. These r e s u l t s s t r o n g l y support the suggestion that the 110 d i f f e r e n c e s h a v e a g e n e t i c b a s i s . T h i s b e i n g t h e c a s e , o n e w o u l d e x p e c t t h e d i f f e r e n t b e h a v i o u r s t o b e t h e p r o d u c t o f n a t u r a l s e l e c t i o n a n d t h u s h a v e s o m e a d a p t i v e s i g n i f i c a n c e t o t h e f i s h i n t h e i r e n v i r o n m e n t s . I n o r d e r t o b e a d a p t i v e , t h e b e h a v i o u r a l p a t t e r n s m u s t m a k e t h e o r g a n i s m b e t t e r a b l e t o s u r v i v e a n d r e p r o d u c e a s c o m p a r e d t o o t h e r m e m b e r s o f t h e s a m e s p e c i e s ( W i l s o n , 1 9 7 5 ) . T h e s e c o n d o b j e c t i v e o f t h i s s t u d y w a s t o a t t e m p t t o t e s t t h e h y p o t h e s i s t h a t t h e o b s e r v e d d i f f e r e n c e s i n b e h a v i o u r a r e , i n p a r t , a d a p t a t i o n s t o t h e d i f f e r e n c e s i n t u r b i d i t y o f t h e s t r e a m s i n w h i c h t h e p o p u l a t i o n s n a t u r a l l y o c c u r . O n t h e b a s i s o f t h i s h y p o t h e s i s I p r e d i c t e d t h a t m a l e s f r o m a c l e a r h e a d s t r e a m r i v e r w o u l d b e m o r e s u c c e s s f u l a t m a t i n g t h a n m a l e s f r o m t h e t h e t u r b i d m i d s t r e a m a n d l o w l a n d r i v e r s w h e n p l a c e d i n c o m p e t i t i o n f o r f e m a l e s i n c l e a r w a t e r . T h i s p r e d i c t i o n w a s p a r t i a l l y s u p p o r t e d . H e a d s t r e a m m a l e s w e r e s i g n i f i c a n t l y m o r e s u c c e s s f u l a t m a t i n g w i t h h e a d s t r e a m f e m a l e s i n c l e a r w a t e r t h a n w e r e m i d s t r e a m a n d l o w l a n d m a l e s . T h i s s t r o n g l y s u g g e s t s t h a t h e a d s t r e a m f e m a l e s c h o o s e m a l e s w i t h h i g h d i s p l a y f r e q u e n c y a n d d u r a t i o n . N o s i g n i f i c a n t d i f f e r e n c e s w e r e f o u n d , h o w e v e r , i n t h e m a t i n g s u c c e s s o f h e a d s t r e a m m a l e s a n d m i d s t r e a m m a l e s w h e n c o m p e t i n g f o r m i d s t r e a m f e m a l e s , n o r i n t h e m a t i n g s u c c e s s o f h e a d s t r e a m m a l e s a n d l o w l a n d m a l e s c o m p e t i n g f o r l o w l a n d f e m a l e s . I s u g g e s t e d t h a t t h e h i g h l e v e l o f c o h e s i o n i d e n t i f i e d i n l o w l a n d a n d m i d s t r e a m p o p u l a t i o n s m a y h a v e r e s u l t e d i n g r e a t e r s u c c e s s i n m a t i n g f o r l o w l a n d a n d m i d s t r e a m m a l e s t h r o u g h e i t h e r d i s p l a y o r t h r u s t i n g . A s e c o n d p r e d i c t i o n b a s e d o n t h e h y p o t h e s i s t h a t t h e I l l o b s e r v e d b e h a v i o u r a l d i f f e r e n c e s b e t w e e n p o p u l a t i o n s w e r e , i n p a r t , a d a p t a t i o n s t o t u r b i d i t y , w a s t h a t m a l e s f r o m t h e t u r b i d l o w l a n d r i v e r w o u l d b e m o r e s u c c e s s f u l i n m a t i n g w i t h f e m a l e s w h e n p l a c e d i n c o m p e t i t i o n w i t h C l e a r w a t e r h e a d s t r e a m males i n t u r b i d w a t e r . L o w l a n d m a l e s w e r e s i g n i f i c a n t l y m o r e s u c c e s s f u l i n m a t i n g w i t h l o w l a n d f e m a l e s t h a n w e r e h e a d s t r e a m m a l e s . T h i s s t r o n g l y s u g g e s t s t h a t m a l e s e x h i b i t i n g a h i g h f r e q u e n c y o f t h r u s t s i n t h e i r c o u r t s h i p h a v e a n a d a v a n t a g e i n m a t i n g i n t u r b i d w a t e r . N o d i f f e r e n c e s w e r e f o u n d i n t h e n u m b e r o f i n s e m i n a t i o n s a t t r i b u t a b l e t o t h e t w o p o p u l a t i o n s i n h e a d s t r e a m f e m a l e s . H e a d s t r e a m m a l e s w e r e f o u n d , h o w e v e r , t o h a v e b e e n m o r e s u c c e s s f u l i n t e r m s o f t o t a l s p e r m c o n t r i b u t e d t o h e a d s t r e a m f e m a l e s . T h e s e r e s u l t s a r e c o n s i s t e n t w i t h t h e s u g g e s t i o n t h a t f e m a l e p r e f e r e n c e f o r m a l e s o n t h e b a s i s o f c o l o u r a n d / o r m a l e p r e f e r e n c e f o r f e m a l e s o f t h e b a s i s o f a p h e r o m o n e c o n t r i b u t e t o m a t i n g s u c c e s s i n h e a d s t r e a m p o p u l a t i o n s . I n c o m p a r i n g t h e r e s u l t s o f t h e c l e a r w a t e r a n d t u r b i d w a t e r e x p e r i m e n t s , t h e o v e r a l l m a t i n g s u c c e s s o f h e a d s t r e a m m a l e s i n c r e a s e d i n c l e a r w a t e r a s c o m p a r e d t o t u r b i d w a t e r . C o n v e r s e l y , t h e o v e r a l l m a t i n g s u c c e s s o f l o w l a n d m a l e s i n c r e a s e d i n t u r b i d w a t e r a s c o m p a r e d t o c l e a r w a t e r . T h e p r e d i c t i o n t h a t m a l e s f r o m t w o t u r b i d w a t e r p o p u l a t i o n s e x h i b i t i n g s i m i l a r c o u r t s h i p s t r a t e g i e s w o u l d n o t e x i b i t d i f f e r e n c e s i n m a t i n g s u c c e s s w h e n p l a c e d i n c o m p e t i t i o n f o r f e m a l e s o f t h o s e p o p u l a t i o n s w a s s u p p o r t e d . A s i m i l a r p r e d i c t i o n f o r b e h a v i o u r a l l y s i m i l a r m a l e s f r o m t w o c l e a r h e a d s t r e a m r i v e r s w a s n o t s u p p o r t e d . I n t h i s 112 experiment, males of each headstream population were s i g n i f i c a n t l y more s u c c e s s f u l at mating w i t h females of t h e i r own populations. These r e s u l t s suggest that the responsiveness of females to the high d i s p l a y frequency and du r a t i o n i n males, while being an important component of mate choice i n headstream popu l a t i o n s , i s at the same time not the only f a c t o r . High success r a t e s of b e h a v i o u r a l l y s i m i l a r males with females from t h e i r own populations suggests t h a t e i t h e r females may be choosing males on the b a s i s of a non-behavioural c h a r a c t e r i s t i c such as colour (Haskins, et a l . , 1961; Endler, 1978, 1980) or p o s s i b l y s i z e ( L i l e y and Seghers, 1975) or that males are a c t i v e l y choosing females on the b a s i s of a population s p e c i f i c pheromone (Crow and L i l e y , 197 9; Crow, 1981). The r e s u l t s of t h i s study c l e a r l y i l l u s t r a t e that a l t e r i n g t u r b i d i t y has a d i r e c t e f f e c t on the mating success of guppies. Under c l e a r water c o n d i t i o n s , males from c l e a r water r i v e r s are more s u c c e s s f u l at mating w i t h females from c l e a r water r i v e r s than are males from t u r b i d r i v e r s . Under t u r b i d water c o n d i t i o n s , on the other hand, males from t u r b i d r i v e r s are more s u c c e s s f u l than males from c l e a r water r i v e r s at mating with females from t u r b i d r i v e r s . Observations of guppies i n the f i e l d and i n the l a b o r a t o r y have confirmed that males from the two types of r i v e r s d i f f e r i n t h e i r c o u r t s h i p behaviour and that these d i f f e r e n c e s are i n part g e n e t i c a l l y determined. These r e s u l t s are c o n s i s t e n t with the hypothesis that high frequencies and durations of d i s p l a y i n the c o u r t s h i p of males from c l e a r water headstreams are adaptations to c l e a r water. The r e s u l t s are a l s o c o n s i s t e n t with the hypothesis that the 113 high frequencies of t h r u s t s i n the c o u r t s h i p behaviour of males from t u r b i d lowland r i v e r s are adaptations to t u r b i d water. My r e s u l t s a l s o suggest that male c o u r t s h i p behaviour i s not the only f a c t o r operative i n i n s u r i n g mating success i n these populations. Some evidence has been found to suggest that female preference for males on the b a s i s of colour or s i z e may be, i n p a r t , an adaptation to c l e a r water c o n d i t i o n s . Grant (1963) s t a t e d that an organism must be adapted to i t s t o t a l environment. This i m p l i e s t h a t the observed behavioural and morphological c h a r a c t e r i s t i c s of an organism are 'responses* to a number of s e l e c t i o n pressures, both b i o t i c and a b i o t i c . I t i s t h e r e f o r e u n l i k e l y that t u r b i d i t y alone can t o t a l l y e x p l a i n the observed v a r i a b i l i t y i n the behaviour of the guppy. I turn now to consider the evidence for other s e l e c t i o n pressures which have been suggested as i n f l u e n c i n g the behavioural and morphological c h a r a c t e r i s t i c s of t h i s s p e c i e s . Sexual S e l e c t i o n . Brown (1975) defined sexual s e l e c t i o n as the d i f f e r e n t i a l production of progeny by d i f f e r e n t genotypes as a r e s u l t of competitive mating. Darwin (1871) himself i d e n t i f i e d the two mechanisms through which sexual s e l e c t i o n can operate, male competition or female choice. Both these a l t e r n a t i v e s , which are not mutually e x c l u s i v e , have been suggested as having i n f l u e n c e d the morphology and behaviour of the guppy. Noble and C u r t i s (1935) , Breder and Coates (1935) and Haskins and Haskins (1949; 1950) have a l l suggested that male c o l o u r a t i o n serves as a warning s i g n a l to other males, rather 114 than as a mechanism for female choice. Both G a n d o l f i (1971) and G o r l i c k (1976) concluded from t h e i r r e s u l t s t h a t dominance h i e r a r c h i e s e x i s t i n l a b o r a t o r y populations of guppies and that the dominant males i n these h i e r a r c h i e s were res p o n s i b l e f o r most of the progeny i n these populations. G o r l i c k suggested that s i z e , sex and aggression played an important part i n i n f l u e n c i n g these h i e r a r c h i e s . Gandolfi on the other hand suggested that female choice on the b a s i s of colour i s the mechanism through which dominant males ensure mating success. While both B a l l i n (1973) and Crow (1981) observed aggression i n n a t u r a l populations i n the l a b o r a t o r y , observations of populations i n t h e i r n a t u r a l environments by both Farr (1975) and myself suggest that male aggression toward other males or females i s almost non-existent i n the w i l d . In competitive mating experiments w i t h inbred stocks of guppies, Farr (1980) found aggression to decrease reproductive success i n males. These r e s u l t s leave i n question the r o l e intermale competition plays i n sexual s e l e c t i o n i n the guppy. Endler (1980) concluded from a s e r i e s of experiments i n which n a t u r a l populations v a r y i n g i n colour patterns were subjected to varying i n t e n s i t i e s of predation i n a number of experimental environments t h a t guppy colour patterns are a f f e c t e d by a balance between sexual s e l e c t i o n and s e l e c t i o n fo r c r y p s i s by predators i n a p a r t i c u l a r environment. In the absence of pr e d a t i o n , Endler found colour patterns i n male guppies to mismatch the background substrate i n terms of b r i g h t n e s s , patch s i z e and colour frequencies. He suggested that inter-male competition and frequency-dependant mating were 115 the mechanisms selecting for t h i s mismatch. Mating a number of inbred stocks varying i n behavioural and morphological c h a r a c t e r i s t i c s , Farr (1980) concluded that high display rate i n males was the prime determinant of reproductive success. Farr assessed the broods from these matings and further concluded that males respond to the display rate of a competitor and that colour only affected reproductive success i f there were no overriding behavioural differences. My results agree with those of Farr (1980) i n that those males from natural populations exhibiting high frequencies and durations of display have greater mating success with females of the same population i n clear water than do males exhibiting lower frequencies and durations of display. These results support the hypothesis that in clear water headstreams sexual selection i n the form of female choice may select for the high frequencies and durations of display. In the absence of behavioural difference I also found evidence that colour patterns or size may e f f e c t female choice i n clear water. Predation Predation has been correlated with behavioural and morphological c h a r a c t e r i s t i c s i n a variety of species of f i s h (see Worthington, 1937; Hoogland, et a l . , 1956; Lowe-McConnell, 1959; 1975; Fryer, 1959; Jackson, 1961; Gross, 1978). In the guppy, the suggestion that male colour patterns represent a balance between sexual selection (favouring bright) and predation (favouring dull) was f i r s t presented by Haskins et a l . , (1961). Endler (1978) predicted that the colour patterns of male guppies at a p a r t i c u l a r place are inconspicuous or 116 c r y p t i c i f they approximate the background i n colour patch size, brightness and colour d i s t r i b u t i o n . He suggested that colour patterns could be predicted based on the i n t e n s i t y of predation i n any given r i v e r . He tested t h i s prediction i n his (1980) study mentioned above. Placing guppies i n a variety of environments modeling natural situations i n terms of substrate background and predator pressure, he tested the effects of predation on colour by censusing a number of generations of f i s h for number, size and position of spots, colour and body siz e . He found guppies to be less conspicuous at high predator as opposed to low predator i n t e n s i t i e s and found a good background matching to colour patterns, that i s , course gravel matched to large spots and fine gravel matching small spots i n those environments i n which predation was most intense. Farr (197 8) observed male courtship behaviour i n a number of populations of guppies i n r i v e r s i n Trinidad and found that high display rate was c h a r a c t e r i s t i c of populations l i v i n g i n r i v e r s with large or no predators while low display rate was c h a r a c t e r i s t i c of populations l i v i n g i n r i v e r s i n which Rivulus  h a r t i i was the only predator. My observations of guppies i n the N and P r i v e r s (IL*. h a r t i i present) and the LA and G r i v e r s (large predators present) furnished results which were opposite to those of Farr. The reasons for t h i s discrepancy are unknown and d i f f i c u l t to trace since l i t t l e i s stated i n the paper concerning his study s i t e s . He observed 50 males i n each river for unspecified periods of time and does not indicate whether the data r e f l e c t behaviour i n one location or i n a variety of microhabitats i n each r i v e r . Other laboratory studies dealing 117 with populations from r i v e r s containing large predators and other populations from r i v e r s containing only R. h a r t i i ( B a l l i n , 1973, Crow, 1981) found results similar to mine. I suggest that predation i s a major factor, along with t u r b i d i t y , balancing sexual selection i n the predator r i c h , turbid lowland r i v e r s . Courtship behaviors which make the male less obvious to predators would l i k e l y be favoured i n the lowland r i v e r s , namely low display frequency and duration and the thrust strategy. L i l e y and Seghers (1975) found Rivulus, which as mentioned e a r l i e r , exists i n headwater streams, to prey s e l e c t i v e l y on smaller guppies while the large lowland predators Crenicichla and Hoplias were unselective or preyed on larger guppies. They suggested that size selective predation may be one of the factors involved i n determining interpopulation v a r i a t i o n i n si z e . The f i s h used i n t h i s study from r i v e r s i n which Crenicichla and Hoplias were present (G and LA) were generally smaller than those from r i v e r s i n which only Rivulus was present (N and P, see Appendix D). Seghers (1970; 1973; 1974) investigated the behavioral 'responses' of natural populations to predation. He found that populations subject to high predation pressures were more cohesive than those i n which only Rivulus was a predator. These observations are substantiated by my estimates of cohesion i n the f i e l d . He presented wild and laboratory raised 'schoolers' (Lower Aripo and Guayamare) and 'non-schoolers' (Naranjo, Paria) in equal numbers to large predators and found them to take s i g n i f i c a n t l y more non-schoolers than schoolers. He 118 r e l a t e d these f i n d i n g s to a l a r g e a n t i - p r e d a t o r complex of adaptions i n c l u d i n g r e a c t i o n d i s t a n c e , alarm thr e s h o l d and m i c r o h a b i t a t s e l e c t i o n . The responses i n terms of 'schooling' might a l s o have been i n f l u e n c e d by the e f f e c t s of t u r b i d i t y on c o u r t s h i p behaviour. Stream V e l o c i t y The e f f e c t s of stream v e l o c i t y on behaviour has been i n v e s t i g a t e d by Crow (1981). As i n t h i s study, guppies from lowland r i v e r s were found to e x h i b i t greater cohesion, a greater frequency of t h r u s t s and a lower frequency of d i s p l a y s than headstream populations. He obtained s i m i l a r r e s u l t s i n w i l d and l a b o r a t o r y r a i s e d f i s h . Lowland guppies were found to be l e s s aggressive and more adherent to the surface area of stream tanks than those from headstreams. Stream v e l o c i t y was a l s o found to have a d i r e c t e f f e c t on c o u r t s h i p behaviour of males, decreasing the frequency of both sigmoid d i s p l a y s and gonopodial swings as v e l o c i t y increased. Males from headstream r i v e r s were found b e t t e r able to maintain the frequency and d u r a t i o n of d i s p l a y i n f a s t water than were lowland males. Crow (1981) concluded that c o u r t s h i p behaviour was t h e r e f o r e a l s o , i n p a r t , adapted to stream v e l o c i t y . Crow found males from two i s o l a t e d r i v e r s to d i s p l a y s e l e c t i v e l y to females of t h e i r own populations. Results of my study, using the same populations, i n d i c a t e that a s s o r t a t i v e mating takes place between these populations. The d i s c r i m i n a t i o n may be based on the male preference i d e n t i f i e d by Crow (1981) and B a l l i n (1973) (possibly on the' b a s i s of a species s p e c i f i c pheromone) or female preference based on 119 colour p a t t e r n . Temperature L i l e y and Seghers (1975) found temperature to have an e f f e c t on the s i z e of a d u l t male guppies. Males of both headstream and lowland r i v e r s grew l a r g e r under a 23 °C regime than those under a 28 °C regime, although i n both temperatures headstream Naranjo males grew l a r g e r than lowland Guayamare males. There were no s i g n i f i c a n t d i f f e r e n c e s found i n the e f f e c t s on females of these stocks. The authors concluded that s i z e d i f f e r e n c e s were i n part due to genetic d i f f e r e n c e s . These studi e s and my own research lead me to conclude that a number of both b i o t i c and a b i o t i c s e l e c t i o n pressures have co n t r i b u t e d to the observed v a r i a t i o n i n the behaviour and morphology of guppies i n the w i l d . Sexual s e l e c t i o n i n the form of female choice has l i k e l y favoured the e v o l u t i o n of l a r g e , b r i g h t l y coloured males e x h i b i t i n g vigorous c o u r t s h i p d i s p l a y s i n c l e a r water headstream r i v e r s . S e l e c t i v e predation by Rivulu s on smaller guppies i s l i k e l y to have c o n t r i b u t e d to the l a r g e s i z e of f i s h i n these r i v e r s as has low temperature, the l a t t e r e s p e c i a l l y i n males. Stream v e l o c i t y has presumably i n t e r a c t e d to set an upper l i m i t on the frequency and d u r a t i o n of c o u r t s h i p d i s p l a y . Predation i s seen as a major s e l e c t i v e f a c t o r r e s p o n s i b l e the l e s s conspicuous colour patterns observed i n male guppies i n lowland r i v e r s . Predation and t u r b i d i t y may also have i n t e r a c t e d to s e l e c t for a l e s s v i s u a l c o u r t s h i p strategy i n males, one r e l y i n g l e s s on the conspicuous d i s p l a y strategy and more on the non-visual t h r u s t s t r a t e g y . Schooling, or cohesiveness, and preference f o r the 120 s u r f a c e a n d e d g e s o f r i v e r s w o u l d a l s o b e f a v o u r e d i n t h e s e l o w l a n d r i v e r s , b o t h a s a n t i - p r e d a t o r s t r a t e g i e s a n d t o e n s u r e m a t i n g u n d e r t u r b i d w a t e r c o n d i t i o n s . T h e s m a l l e r s i z e o f l o w l a n d f i s h m a y h a v e b e e n i n f l u e n c e d b y b o t h p r e d a t o r p r e f e r e n c e a n d t e m p e r a t u r e . T h e r e v i e w a l s o i l l u s t r a t e s t h a t m o s t o f t h e s t u d i e s c o n d u c t e d t o d a t e h a v e c o n c e n t r a t e d o n o n e s e l e c t i v e a g e n t a n d f e w o f e i t h e r b e h a v i o u r a l o r m o r p h o l o g i c a l c h a r a c t e r i s t i c s . C o n s e q u e n t l y , r e s u l t s a r e o b t a i n e d i n i s o l a t i o n f r o m o t h e r f a c t o r s a n d c h a r a c t e r i s t i c s , a n d i n t e g r a t i o n b e c o m e s d i f f i c u l t a n d s o m e t i m e s c o n t r a d i c t o r y . A l s o , s t u d i e s u s i n g i n b r e d l i n e s o f g u p p i e s l e a v e s o m e d o u b t a s t o t h e i r a p p l i c a t i o n t o t h e n a t u r a l s i t u a t i o n . A f u r t h e r c o m p l i c a t i o n h a s i n v o l v e d d i s t i n g u i s h i n g b e t w e e n p h e n o t y p i c a n d g e n e t i c e f f e c t s . W h i l e i t w i l l r e m a i n i m p o s s i b l e t o a s s e s s a l l t h e p o s s i b l e s e l e c t i v e f a c t o r s a f f e c t i n g t h e g u p p y a n d d e t e r m i n i n g t h e i r e x a c t c o n t r i b u t i o n s t o b e h a v i o u r a n d m o r p h o l o g y , i t s h o u l d b e p o s s i b l e t o d e s i g n l o n g t e r m e x p e r i m e n t s s u c h a s t h o s e c o n d u c t e d b y E n d l e r ( 1 9 8 0 ) i n w h i c h a v a r i e t y o f b i o t i c a n d a b i o t i c c o n d i t i o n s w e r e s i m u l a t e d i n e x p e r i m e n t a l e n v i r o n m e n t s . U s i n g n a t u r a l p o p u l a t i o n s , t h e a c t u a l s e l e c t i v e e f f e c t s o f t h e c o n d i t i o n s c o u l d t h e n b e a s s e s s e d d i r e c t l y , b y o b s e r v i n g f o r c h a n g e s i n a n u m b e r o f m o r p h o l o g i c a l a n d b e h a v i o u r a l c h a r a c t e r i s t i c s o v e r s e v e r a l g e n e r a t i o n s . D i r e c t a n d i n t e r a c t i o n e f f e c t s c o u l d t h e n b e d e t e r m i n e d t h r o u g h t h e u s e o f a m u l t i v a r i a t e s c h e m e . I d e a l l y , t h e s e t y p e s o f e x p e r i m e n t s c o u l d b e c a r r i e d o u t i n t h e n a t u r a l e n v i r o n m e n t s . S t o c k s c o u l d b e c o l l e c t e d a n d 121 placed i n cages similar to the ones used i n t h i s study. Populations from headstreams could, for example, be placed i n the Guayamare and the lowland f i s h in the Naranjo, both with and without representative predators i n the cages. The stocks could then be allowed to breed and the i r behavioural and morphological c h a r a c t e r i s t i c s assessed over a number of generations. The only serious l i m i t a t i o n to conducting studies i n the natural environment i s a p r a c t i c a l one, human intervention. 122 LITERATURE CITED Affleck, R.J. 1960. 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Hydrogr. 3_5_: 304- 317 . 128 129 APPENDIX A FIGURE 14 T h e m e a n n u m b e r o f m a l e s ( M ) , f e m a l e s ( F ) , a n d j u v e n i l e s ( J ) i n 2 5 0 0 cm.2 a r e a s i n t h e f o u r r i v e r s . 95% c o n f i d e n c e l i m i t s o f t h e m e a n s a r e i n d i c a t e d . T h e d a t a w a s o b t a i n e d a s d e s c r i b e d i n C h a p t e r 3 . 20 — , CM O o o OJ £ 10 CQ ± M F J NARANJO T M F J PARIA M. F J LOWER ARIPO .— 20 10 00 o M F J GUAYAMARE Characteristic Number of fish per 2500 cm Distance between fish (cm) Depth of water (cm) Distance to shore (cm) Water velocity (m/sec) Number of sigmoid displays/3 min Number of gonopodial thrusts/3 min Number of copulation attempts/3 min Duration of sigmoid display (sec) APPENDIX B Statist ical values for Bart le t t s 1 , ANOVA and Scheffes' Tests on the f ie ld observations of populations in the four r ivers . BARTLETT'S X 2 ANOVA df SCHEFFE9 SCHEFFE COMPARISONS13 G-LA G-N G-P LA-N LA-P 3.962 0.266 6.443 3.64 0.199 0.978 4.480 3,64 N-P 5.183 0.159 44.531 3,200 0.0001 3.4117 5.814 11.444 7.135 6.019 1.140 5.250 9.637 0.022 41.099 3,64 0.0001 3.5071 2.906 9.469 8.228 7.204 5.794 1.580 0.0007 3.5071 5.268 6.273 5.001 0.869 0.599 1.569 0.0071 3.5071 1.700 3.906 9.405 2.740 9.556 5.870 10.992 0.012 7.754 3,64 0.0002 3.5071 1.056 4.350 2.195 3.636 1.216 2.631 4.814 0.186 13.964 3,336 0.0001 3.4010 0.191 3.448 4.903 3.872 5.725 2.219 4.199 0.241 18.224 3,336 0.0001 3.4010 3.793 6.584 6.354 3.306 2.649 1.060 4.785 0.197 0.839 3,336 0.4733C - - - - - - -2.273 0.482 13.201 3,536 0.0001 3.3850 0.288 3.414 4.831 3.454 5.235 1.491 a Scheffe F calculated at 0.01 level of significance. b Any comparison value, and thereby the contrast i t represents, that is greater than the Scheffe F value is significant at the 0.01 level of significance (underlined). c The ANOVA^for this comparison was found to be not significant at the 0.01 level of significance, therefore no Scheffe was performed on the data. APPENDIX . C •Statistical values for Bart le t t s 1 , ANOVA and Scheffes' Tests on the laboratory observations of populations from the four r ivers . BARTLETT'S ANOVA SCHEFFE3 SCHEFFE COMPARISONS^ Characteristic x2 F df F G-LA G-N G-P LA-N LA-P N-P displays/10 9 min d 5 , 2 0 8 0 , 1 5 7 3 6 - 6 2 7 3 > 1 1 6 0.0001 3.447 3.865 11.849 9.223 7.981 5.358 2.626 Number of gonopodial thrusts/10 min. 11.272 0.011 11.170 3,116 0.0001 3.447 1.427 3.451 5.439 1.924 4.013 2.089 Number of copulation , n c o n n 0 1 1 C n „ C r C attempts/10 min. 1 , 9 6 3 0 , 5 8 0 0 , 7 3 1 3 ' 1 1 6 0 , 5 3 5 5 " - - - - - - -d i s p f a y ^ s e c ) 1 9 " 1 0 ^ 8 - 8 5 7 ° " ° 5 2 8 0 , 4 3 9 3 ' 9 6 2 0 , 0 0 0 1 3 , 3 7 6 3 , 7 3 0 1 2 - 7 5 0 8 - 5 5 9 H-090 5.633 6.829 sw?ngs/10 m i n ? ^ 1 * 1 5 , 9 2 7 0 , 1 1 5 1 9 , 2 7 8 3 ' 1 1 6 0 , 0 0 0 1 3 , 4 4 7 1 , 2 9 4 5 , 2 4 1 ! - 2 5 6 6-536 0.039 6.496 5^nM rSn] , e 8-443 °-038 1-W2 3'116 ° - 3 6 « C - " " - " a Scheffe F calculated at 0.01 level of significance. b Any comparison value, and thereby the contrast i t represents, that is greater than the Scheffe F value is significant at the 0.01 level of significance (underlined). c The ANOVA^for this comparison was found to be not significant at the 0.01 level of significance, therefore no Scheffe was performed on the data. CO 133 APPENDIX D MEAN TOTAL LENGTH OF GUPPIES OF ALL POPULATIONS USED IN OBSERVATIONS AND EXPERIMENTS. STANDARD ERRORS IN BRACKETS. Behavioural Observations (Chapter 3 ) . Population Guayamare Lower Aripo Naranj o P a r i a Males 14.513 (0.165) 15.240 1(0.132) 17.890 (0.234) 15.927 (1.952) Females 17.371 (0.597) 19.367 (0.278) 20.486 (0.414) 19.633 (0.451) B. Competitive Mating i n Clear Water (Chapter 4). Population Guayamare Lower Aripo Naranjo P a r i a Males 14.540 (0.719) 14.669 (0.144) 17.878 (0.258) 17.613 (0.212) Females 18.399 (0.211) 19.182 (0.288) 20.551 19.606 (0.317) (0.361) C. Competitive Mating i n Turbid Water (Chapter 5). Population Males Females Guayamare Naranj o 14.708 (0.127) 18.118 (0.177) 18.636 20.849 (0.284) (0.535) 134 APPENDIX E FORMULAS FOR STAINS AQUEOUS EOSIN Y Dissolve 5 gm of Eosin Y i n 1000 ml d i s t i l l e d water. MAYER'S HEMATOXYLIN Dissolve 1 gm of Hematoxylin i n 1000 ml d i s t i l l e d water Then add 0.2 gm sodium iodate and 50 gm aluminum s u l f a t S t i r . 

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