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Reproductive isolation between two co-existing populations of stickleback (Gasterosteus) in Enos Lake,… Ridgway, Mark S. 1982

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REPRODUCTIVE ISOLATION BETWEEN TWO CO-EXISTING POPULATIONS OF STICKLEBACK (GASTEROSTEUS) IN ENOS LAKE, VANCOUVER ISLAND by MARK S. RIDGWAY B.Sc., University of Miami 1978 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept this thesis as conforming to the required standard BRITISH COLUMBIA 1 982 THE UNIVERSITY OF March c) Mark S. Ridgway, 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 The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (.3/81) i i ABSTRACT M o r p h o l o g i c a l , b i o c h e m i c a l , and t r o p h i c evidence from other s t u d i e s i n d i c a t e that two p o p u l a t i o n s of s t i c k l e b a c k (Gasterosteus) c o - e x i s t i n Enos Lake, Vancouver I s l a n d . One p o p u l a t i o n i s l i m n e t i c and the other i s benthic (the names a l l u d e to t h e i r f o r a g i n g behaviour and use of space). In t h i s study, f i e l d o b s e r v a t i o n s , mate s e l e c t i o n experiments, and c o u r t s h i p behaviour experiments were conducted to determine i f the two s t i c k l e b a c k p o p u l a t i o n s are r e p r o d u c t i v e l y i s o l a t e d . When breeding, l i m n e t i c males develop red t h r o a t s whereas benthic males become un i f o r m l y b l a c k . In the f i e l d l i m n e t i c and benthic males nest i n d i f f e r e n t h a b i t a t s . Despite t h i s d i f f e r e n c e , r e p r o d u c t i v e males and females of each p o p u l a t i o n encountered each other, but i n the few cases where c o u r t s h i p occurred, i t never went beyond the i n i t i a l stages of the l e a d -f o l l o w sequence. Apparently b e h a v i o u r a l d i f f e r e n c e s i n c o u r t s h i p c o n t r i b u t e d to the break o f f of these n a t u r a l c o u r t s h i p s . In mate s e l e c t i o n experiments, males and females of each p o p u l a t i o n p r e f e r r e d mates from t h e i r own p o p u l a t i o n . In the c o u r t s h i p experiments, b e h a v i o u r a l d i f f e r e n c e s between l i m n e t i c s and b e n t h i c s were found to be g r e a t e s t i n the e a r l y stages of c o u r t s h i p . With females from t h e i r own p o p u l a t i o n , benthic males were more a g g r e s s i v e in t h e i r approach and l e a d i n g sequences than l i m n e t i c males. In the beginning of the l e a d -f o l l o w phase, benthic females tend to p o s i t i o n themselves above benthic males whereas l i m n e t i c females tend to p o s i t i o n themselves a l o n g s i d e l i m n e t i c males. Male c o u r t s h i p behaviour was sometimes dependent on the phenotype of the female courted; benthic males b i t and chased l i m n e t i c females whereas, l i m n e t i c males b i t and l e d benthic females i n a meandering path to the nest. Since the mate ch o i c e experiments i n d i c a t e d t o t a l p o s i t i v e a s s o r t a t i v e mating between l i m n e t i c s and b e n t h i c s , i t i s l i k e l y that the b e h a v i o u r a l d i f f e r e n c e s found i n c o u r t s h i p behaviour c o n t r i b u t e to r e p r o d u c t i v e i s o l a t i o n between the l i m n e t i c and benthic s t i c k l e b a c k s i n Enos Lake. i i i TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES . i v LIST OF FIGURES v ACKNOWLEDGEMENTS v i General I n t r o d u c t i o n 1 General Methods 4 C o l l e c t i o n and Maintenance of F i s h 4 Observation and Recording Procedures 5 Male and Female Behaviours 9 F i e l d Observations 13 I n t r o d u c t i o n 13 Methods 13 Re s u l t s 15 Nesting Limnetic Males 15 Nesting Benthic Males 16 S e l e c t i v e Trapping 18 Conclusion 20 Mate S e l e c t i o n Experiments 21 I n t r o d u c t i o n 21 Methods 22 Res u l t s 23 Female Mate S e l e c t i o n 23 Male Mate S e l e c t i o n 24 Conclusion 25 Co u r t s h i p Experiments 26 I n t r o d u c t i o n 26 Methods 27 Re s u l t s 30 Male Approach and Female Response 32 Male Lead and Female Follow 37 Male and Female at the Nest 49 Male Swim to the Nest Alone 54 Conclusion 54 D i s c u s s i o n 57 References 64 Appendix i 69 Appendix a' 70 Appendix 3? 71 Appendix 4 7 2 Appendix 5 73 iv LIST OF TABLES Table 1. Number of males captured in open areas and dense vegetation 18 Table 2. Number of courtship t r i a l s ending with nest entry by the female 28 Table 3. Male and female courtship behaviours 29 Table 4. Number of courtship cycles 32 Table 5. Summary of male approach and female response 33 Table 6. Mean number of bites/minute, zig-zag bouts/minute, and female swim to male bouts/minute 34 Table 7. Summary of positive female responses to three types of male approach 35 Table 8. Summary of positive and negative lead-follow sequences 44 Table 9. Summary of female position r e l a t i v e to the male at the start of lead-follow sequences 45 Table 10. Number of di r e c t or meandering male leads 46 Table 11. Male destination when male and female break-away from lead-follow sequences 48 Table 12. Male bites during unsuccessful lead-follow sequences 49 Table 13. Mean frequency per courtship cycle of females in or out of position while at the nest with the male 51 Table 14. Mean frequency per courtship cycle of female tryi n g the nest entrance 52 Table 15. Mean length of time between female a r r i v a l at the nest and trying the nest entrance 53 Table 16. Number of times males l e f t females, for their nests after approach or lead-follow sequences 55 V LIST OF FIGURES Fi g u r e 1. Photographs of l i m n e t i c and benthic s t i c k l e b a c k s 6 F i g u r e 2. Cumulative frequency d i s t r i b u t i o n s of the d u r a t i o n s of male approach and p o s i t i v e female response f o r l i m n e t i c males c o u r t i n g l i m n e t i c and benthic females F i g u r e 3. Cumulative frequency d i s t r i b u t i o n s of the d u r a t i o n s of male approach and p o s i t i v e female response for l i m n e t i c and benthic males c o u r t i n g benthic females 40 F i g u r e 4. Cumulative frequency d i s t r i b u t i o n s of the d u r a t i o n s of male approach and p o s i t i v e female response f o r males c o u r t i n g females from t h e i r own p o p u l a t i o n ... 42 v i ACKNOWLEDGEMENTS I would l i k e to thank my supervisor, Dr. J. D. McPhail, for his encouragement and advice throughout t h i s study. Our discussions greatly enhanced my focus on the Enos Lake-"scenario", my approach to the problem, and my understanding of f i s h behaviour and ecology in general. As a result, more questions have been created than answered. Also, I would l i k e to thank Drs. L i l e y and Scudder for reviewing the thesis and providing many useful comments. A special thanks to Paul Bentzen; fellow b i o l o g i s t and f r i e n d . He provided advice on every aspect of this study as well as the photographs for Figure 1. Together, helping each other with our respective f i e l d work proved convenient, fun, and fuel for a l o t of s t o r i e s . Thanks must also go to Steve Campana, Dennis Lassuy, and Marvin Rosenau for f i e l d assistance at various times. The "temple" w i l l never be the same. Marvin and I also translated horrendus computer programs. Dave Z i t t i n (Wizard of UNIX) helped with programming, interpreted manuals, and provided computing help whenever I needed to be bailed out. Dr. Lee Gass helped formulate my understanding of animal behaviour in the course of our discussions. His influence is not immediately obvious in the thesis but i s nonetheless present. Mr. Neil Scott kindly permitted us to work at Enos Lake. I would l i k e to thank him for l e t t i n g us stay at his beach cabin during the winter months. Without that cabin, there would have been a lot of miserable f i e l d t r i p s and long nights in the Arlington. F i n a l l y , a special thanks to my family who provided moral and material support when the going got tough. 1 GENERAL INTRODUCTION Freshwater p o p u l a t i o n s of the t h r e e s p i n e d s t i c k l e b a c k (Gasterosteus a c u l e a t u s ) occur throughout the s p e c i e s north temperate range. There i s c o n s i d e r a b l e i n t e r p o p u l a t i o n v a r i a b i l i t y (Hagen, 1967; M i l l e r and Hubbs, 1969; Moodie, 1972a; B e l l , 1981; B e l l and Ric h k i n d , 1981), and some of the morphological d i f f e r e n c e s between p o p u l a t i o n s are known to be the r e s u l t of n a t u r a l s e l e c t i o n (Hagen and G i l b e r t s o n , 1973; Moodie et a l , 1973; B e l l and Haglund, 1978; Gross, 1978). U s u a l l y these m o r p h o l o g i c a l l y d i f f e r e n t p o p u l a t i o n s are a l l o p a t r i c (Munzing, 1963; Hagen and G i l b e r t s o n , 1972; B e l l , 1976; Gross, 1977), but o c c a s i o n a l l y d i v e r g e n t p o p u l a t i o n s come in c o n t a c t (Hagen, 1967; McPhail, 1969; Moodie, 1972a; Semler, 1971; Larsen, 1976). In the P a c i f i c Northwest such contact i s u s u a l l y p a r a p a t r i c (that i s , the p o p u l a t i o n s abut but do not overlap ) (Hagen, 1967; McPhail, 1969; Moodie, 1972a,b). Recently, however, sympatric m o r p h o l o g i c a l l y d i v e r g e n t p o p u l a t i o n s of s t i c k l e b a c k s have been found in a few small c o a s t a l lakes in B r i t i s h Columbia. Enos Lake (49°17'N 124°09'W), near Nanoose Bay on Vancouver I s l a n d , i s such a la k e . Biochemical ( W i t h l e r , 1980), morphological (McPhail, i n prep . ) , and t r o p h i c (Bentzen, i n prep.) evidence i n d i c a t e s that two p o p u l a t i o n s of s t i c k l e b a c k s occur in Enos Lake. One p o p u l a t i o n i s p e l a g i c , countershaded ( s i l v e r and b l u e ) , and feeds l a r g e l y on plankton. The other p o p u l a t i o n has a l a r g e r body s i z e , i s mottled black and olive-brown, and feeds l a r g e l y 2 on benthic prey. For convenience, the two populations are referred to as "limnetics" and "benthics". The names indicate differences in feeding habits and the use of space by the two populations. Breeding limnetic males, develop the red throat colouration t y p i c a l of Gasterosteus, whereas breeding benthic males become uniformly black. Limnetic males and females have slender bodies, narrow snouts, and large protrusive eyes. Benthic males and females have deep bodies, short snouts, and non-protrusive eyes. Enos Lake sticklebacks that are intermediate between the two populations are rare (Bentzen et a l , in prep.), but laboratory hybrids are f u l l y f e r t i l e (McPhail, in prep). The . purpose of t h i s study is to examine hypotheses concerning the maintenance of sympatric . limnetic and benthic populations in Enos Lake. Since the limnetic-benthic divergence in Enos Lake appears to be stable (at least for the last five years) and hybrids are i n t e r f e r t i l e , the divergence must be maintained either by pos i t i v e assortative mating or intense selection against hybrids. Thus, one hypothesis i s that mating is random and the divergence is maintained by intense selection against hybrids. The alternate hypothesis is that mating is non-random (that i s , limnetics and benthics recognize each other and p r e f e r e n t i a l l y mate with their own form). Under this hypothesis divergence i s maintained by positive assortative mating; however, there may s t i l l be intense selection against hybrids. If non-random mating occurs in Enos Lake sticklebacks, i t should be possible to demonstrate the role of behaviour in the 3 phenomenon. The f o l l o w i n g methods were used to i n v e s t i g a t e the mating system i n Enos Lake s t i c k l e b a c k s . F i r s t , p r e l i m i n a r y f i e l d o b s e r v a t i o n s were made on n e s t i n g l i m n e t i c and benthic males to determine i f males encountered g r a v i d females of both types i n the f i e l d . Second, l a b o r a t o r y mate s e l e c t i o n experiments were conducted to determine i f males and females p r e f e r mates of t h e i r own p o p u l a t i o n . T h i r d , the c o u r t s h i p behaviour of the two p o p u l a t i o n s were compared to determine i f there are d i f f e r e n c e s i n t h e i r r e p r o d u c t i v e behaviour. 4 GENERAL METHODS C o l l e c t i o n and M a i n t e n a n c e o f F i sh S t i c k l e b a c k s were c o l l e c t e d from Enos Lake from F e b r u a r y 1979 t h r o u g h t o A p r i l 1981. They were t a k e n t o t h e l a b o r a t o r y and s e p a r a t e d i n t o l i m n e t i c s and b e n t h i c s on t h e b a s i s of t h e i r g e n e r a l a p p e a r a n c e ( F i g u r e 1 ) . I n d i v i d u a l s from e a c h p o p u l a t i o n were kept s e p a r a t e l y i n l a r g e communal t a n k s . As t h e y became t e r r i t o r i a l m a l e s were removed and p l a c e d i n e x p e r i m e n t a l a q u a r i a . E a c h e x p e r i m e n t a l a q u a r i u m was d i v i d e d i n h a l f by a p a r t i t i o n , and t h e b o ttoms of a l l a q u a r i a were c o v e r e d w i t h washed b e a c h g r a v e l . The a q u a r i a were a e r a t e d t h r o u g h u n d e r g r a v e l f i l t r a t i o n . Food c o n s i s t e d of l i v e T u b i f e x , c h o p p e d c o m m e r c i a l s h r i m p , o r a mulched h e r r i n g and c o d m i x t u r e . The l i g h t s c h e d u l e f o r a l l a q u a r i a was 16 h o u r s of l i g h t and 8 h o u r s of d a r k ; t e m p e r a t u r e was u n c o n t r o l l e d and r a n g e d from 13°C t o 20°C. The wooden r e c t a n g u l a r e x p e r i m e n t a l a q u a r i a (280L; i n t e r i o r d i m e n s i o n s , 117cm l o n g ; 57cm wide; 33cm deep) were s e a l e d w i t h f i b e r g l a s s and s t a i n e d a u n i f o r m brown. The f r o n t s were g l a s s and t h e a q u a r i a were p a r t i a l l y d i v i d e d w i t h a s e c t i o n of brown p e r f o r a t e d p l e x i g l a s s 44.5 cm l o n g . D e p e n d i n g on t h e e x p e r i m e n t , th e r e m a i n i n g 15.5 cm o f t h e p a r t i t i o n c o n s i s t e d of a r e m o v a b l e p i e c e of c l e a r or b l a c k p l e x i g l a s s . In mate s e l e c t i o n e x p e r i m e n t s , t h e b l a c k p a r t i t i o n was u s e d f o r male c h o i c e e x p e r i m e n t s , and t h e c l e a r p a r t i t i o n was u s e d f o r f e m a l e c h o i c e 5 experiments. In courtship experiments, the black p a r t i t i o n was used to prevent v i s u a l contact between males when both halves of the aquaria were occupied. The clear p a r t i t i o n was usually in place when only one half of an aquarium was occupied. Detritus and sand f i l l e d p l a s t i c pie pans provided a nesting s i t e and broken clay flower pots or stones provided cover. The coarse •beach gravel on the bottom discouraged males from building anywhere execpt in the pie pans. Nesting material was generously scattered over the gravel. If males were reluctent to build, I induced nest building by adding gravid females in jars to the aquarium. In such cases, I removed the females when the male started to b u i l d . To provide diffuse f i l t e r e d l i g h t , f l o a t i n g duckweed (Lemma) was added to a l l aquaria. Observation and Recording Procedures The experimental aquaria were isolated from outside disturbance by a plywood chamber. Observations were made from behind a black p l a s t i c curtain provided with eye holes. From that position I introduced f i s h and recorded behaviours. As many animals as possible were used in a l l mate selection and courtship experiments. Females were kept in communal tanks and used as they became gravid. After an experiment females were returned to the communal tanks and reused i f they again became gravid. Males with-nests were usually tested once a day and kept for a maximum of 4 weeks; however, since the breeding season is 6 F i g u r e 1. Photographs of l i m n e t i c and benthic s t i c k l e b a c k s . The top f i g u r e i s a photograph of a l i m n e t i c male and female (male on t o p ) . The bottom f i g u r e i s a photograph of a benthic male and female (male on bottom). 7 8 short and r e l a t i v e l y few females are gravid at any one time, some males and females were tested twice in the same day. In such cases, gravid females were never tested twice with the same male in the same day. For a l l f i s h there was a minimum of 3 hours between two tests on a single day. Males were considered ready for testing after they had completed their nest and had exhibited "creeping through" behaviour (Sevenster, 1961). To determine i f females were responsive to courtship, they were pre-tested with a male of their own form. If they gave the head-up display (Wootton 1976; and next section) or swam towards the test male they were judged to be ready for testing. Males used to test female readiness were never used for experiments on the same day. A l l successful courtship sequences, whether in mate selection or courtship experiments, ended with the female entering the nest. Females were prevented from depositing their eggs by quickly nipping their caudal peduncles with long tweezers. This simulated male nipping behaviour and caused the females to leave the nest. Thus courting males were never allowed eggs and did not proceed into their parental phase. Behaviours were recorded on an electronic data c o l l e c t i o n system (Observational Systems' Microprocessor Operated Recording Equipment "MORE"). For data r e t r i e v a l and analysis, the system was interfaced with a PDP11 computer. The MORE i s a hand-held, solidstate recording device. Events are recorded by pressing keys representing d i f f e r e n t behaviours. Since a l l experiments involved releasing f i s h into a tank, there was always a moment 9 of i n a c t i v i t y that allowed me time to p o s i t i o n my hands for r e c o r d i n g . Recording began when the females were r e l e a s e d i n t o the aquarium. Male and Female Behaviours A l a r g e number of behaviours have been d e s c r i b e d for the c o u r t s h i p of the t h r e e s p i n e s t i c k l e b a c k (G. a c u l e a t u s ) . The general p a t t e r n of c o u r t s h i p in Gasterosteus begins with a male approaching a female, the female swimming to the male, the male l e a d i n g the female down to h i s nest, and the male i n d i c a t i n g h i s nest entrance to the female who enters or leaves.1 Most of the c o u r t s h i p behaviours are d e s c r i b e d in Wootton (1976; Chapter 12 and r e f e r e n c e s c i t e d t h e r e i n ) , however, as a guide to readers, a b r i e f d e s c r i p t i o n of each of the behaviours I recorded i s i n c l u d e d below. For r e c o r d i n g purposes, the male was f o c a l animal. The male behaviours are: (1) D i r e c t Approach: Male swims s t r a i g h t to the female; (2) Zig-zag Approach: Male approaches the female i n a s e r i e s of l a t e r a l jumps; (3) B i t e : A general category to i n c l u d e male b i t e s at any p o i n t in the c o u r t s h i p t r i a l ; 10 (4) D i r e c t Lead: Male swims i n a s t r a i g h t path back to h i s nest while the female f o l l o w s i n c l o s e p r o x i m i t y ; (5) Meandering Lead: Male swims in a meandering path ( i . e . , not s t r a i g h t ) back to h i s nest while the female f o l l o w s i n c l o s e p r o x i m i t y ; (6 ) D o r s a l P r i c k : Male p o s i t i o n s himself below and l o n g i t u d i n a l l y to the female, and with h i s d o r s a l s p i n e s , moves s l i g h t l y backwards and up as i f to p r i c k the female's b e l l y ; (7) Show Nest Entrance: With the female i n c l o s e p r o x i m i t y , the male turns on h i s s i d e , and i n a j e r k i n g motion with h i s mouth opening and c l o s i n g , he pokes h i s snout i n t o the nest entrance; (8) Nest Maintenance: A general category to i n c l u d e male nest maintenance behaviours while he i s alone at h i s nest (fanning the nest entrance with h i s p e c t o r a l f i n s ; poking and boring at the nest with h i s snout, and g l u e i n g the nest m a t e r i a l by smoothly swimming over the nest with h i s c l o a c a l region pressed a g a i n s t the nest m a t e r i a l ) ; (9) Creep Through Nest: At the end of nest b u i l d i n g and at times during c o u r t s h i p , the male pushes h i s way through the nest producing a tunnel with a d i s t i n c t entrance and e x i t , and ( 1 0 ) N u l l : Male swims in any d i r e c t i o n or remains motionless i n 11 t h e w a t e r ( i . e . , b e h a v i o u r t h a t can not be p l a c e d i n t h e p r e v i o u s c a t e g o r i e s ) . The f e m a l e b e h a v i o u r s a r e : (1 ) Swim t o M a l e : In r e s p o n s e t o a male's a p p r o a c h , t h e f e m a l e swims t o w a r d s t h e male w i t h h e r head up from a h o r i z o n t a l p o s i t i o n ("head-up"); (2) F o l l o w Above: W h i l e t h e male l e a d s t h e f e m a l e t o t h e n e s t , th e f e m a l e swims i n c l o s e p r o x i m i t y above t h e male and w i t h h e r head s l i g h t l y back f rom t h e m a l e ' s head; (3) F o l l o w A l o n g s i d e : W h i l e t h e male l e a d s t h e f e m a l e t o t h e n e s t , t h e f e m a l e swims i n c l o s e p r o x i m i t y a l o n g s i d e t h e male w i t h h e r head s l i g h t l y back from t h e male ' s head; (4) F o l l o w B e h i n d : W h i l e t h e male l e a d s t h e f e m a l e t o t h e n e s t , t h e f e m a l e f o l l o w s b e h i n d t h e male and a t any d i s t a n c e from him; (5) T r y N e s t E n t r a n c e : In r e s p o n s e t o t h e m a l e ' s show n e s t e n t r a n c e b e h a v i o u r , t h e f e m a l e pokes h e r s n o u t i n t o t h e n e s t e n t r a n c e and e i t h e r e n t e r s t h e n e s t or p u l l s o u t ; (6) E n t e r N e s t : A f t e r t r y i n g t h e n e s t e n t r a n c e , t h e f e m a l e p u s h e s h e r way i n t o t h e n e s t , and 12 (7) Leave Nest Area: Female leaves the nest area without e n t e r i n g the nest. Since a l l females tend to o r i e n t in a "head-up" p o s i t i o n when c o n f i n e d with a c o u r t i n g male, the female "head-up" p o s i t i o n was not recorded as a d i s t i n c t behaviour. Enos Lake l i m n e t i c and benthic females were never observed to follow below a l e a d i n g male. The Wilcoxon-Mann-Whitney U t e s t , l o g l i k e l i h o o d r a t i o t e s t (G-test) with W i l l i a m ' s c o r r e c t i o n f a c t o r , and the Kolmogorov-Smirnov two-sample t e s t were used to analyze the recorded behaviour (Sokal and Rohlf, 1 9 8 1). The n u l l h ypothesis f o r comparisons was that there was no d i f f e r e n c e i n c o u r t s h i p behaviour between l i m n e t i c and be n t h i c forms. 13 FIELD OBSERVATIONS Introduct ion Contact between l i m n e t i c s and b e n t h i c s in nature i s necessary f o r a c t i v e mate choice to occur. Other mechanisms such as r e p r o d u c t i v e s e a s o n a l i t y and e c o l o g i c a l or micro-geographic s e p a r a t i o n c o u l d l e a d to p a s s i v e non-random mating; however, these c o n d i t i o n s t y p i c a l l y are incomplete i s o l a t i n g mechanisms in sympatric north temperate f i s h e s , whereas mate choice i s o f t e n an e f f e c t i v e b a r r i e r to mis-mating (Clark and Keenleyside, 1967; Hagen, 1967; B a r t n i k , 1970; Keenleyside, 1978). Methods F i e l d o b s e r v a t i o n s were made on an aggregation of l i m n e t i c males f o r 135 hours i n l a t e June and e a r l y J u l y , 1980. During t h i s p e r i o d i n d i v i d u a l males were observed each day and t h e i r t e r r i t o r i a l and sexual c o n t a c t s recorded on a pre-designed check sheet. P a r t i c u l a r a t t e n t i o n was p a i d to o c c a s i o n a l sexual encounters between benthic females and l i m n e t i c males. T h i s was done to determine the p o i n t ( i f any) that the p a i r broke o f f c o u r t s h i p . If c o u r t s h i p i n c o m p a t i b i l i t y e x i s t s , the p r e d i c t i o n i s that c o u r t s h i p w i l l be broken o f f e a r l y in the c o u r t s h i p sequence. 1 4 Due t o d i f f i c u l t i e s i n l o c a t i n g b e n t h i c male n e s t s i n 1980, a d d i t i o n a l o b s e r v a t i o n s were made i n Ju n e , 1981. D u r i n g t h i s p e r i o d a s p e c i a l e f f o r t was made t o l o c a t e b e n t h i c n e s t s . T h e s e o b s e r v a t i o n s , combined w i t h a few d a y s i n 1980, p r o v i d e d t h e o n l y f i e l d o b s e r v a t i o n s on n e s t i n g b e n t h i c m a l e s . M a l e s were not i n d i v i d u a l l y marked f o r i d e n t i f i c a t i o n ; however, t h e y were e a s i l y i d e n t i f i e d by t h e i r a r e a of r e s i d e n c e w i t h i n t h e n e s t i n g a g g r e g a t i o n . The t o t a l t i m e s p e n t o b s e r v i n g l i m n e t i c and b e n t h i c m a l e s was o v e r 200 h o u r s . S i n c e b e n t h i c m a l e s were d i f f i c u l t t o l o c a t e , minnow t r a p s p l a c e d i n open a r e a s o r i n v e g e t a t i o n were u s e d t o d e t e r m i n e i f c o n c e a l i n g v e g e t a t i o n was o v e r t h e n e s t s i t e s of l i m n e t i c and b e n t h i c m a l e s . The c o v e r a r o u n d n e s t s was d i v i d e d i n t o two c a t e g o r i e s on t h e b a s i s of t h e amount of c o n c e a l i n g v e g e t a t i o n . N e s t s were c o n s i d e r e d t o be i n t h e open i f c o v e r was a b s e n t f o r a p p r o x i m a t e l y 50 cm a r o u n d t h e n e s t . N e s t s were c o n s i d e r e d c o n c e a l e d i f v e g e t a t i o n was so dense a r o u n d t h e n e s t t h a t s u b s t r a t e and n e s t s were d i f f i c u l t t o l o c a t e . The number of b r i g h t l y c o l o u r e d t e r r i t o r i a l m a les was r e c o r d e d from e a c h t r a p . 1 5 R e s u l t s N e s t i n g L i m n e t i c M a l e s F o r 20 d a y s 3 t o 5 g r a v i d l i m n e t i c f e m a l e s were o b s e r v e d t o move o n t o t h e l i m n e t i c male n e s t i n g a r e a e a c h m o r n i n g . D a t a were no t r e c o r d e d i n t h e a f t e r n o o n s s i n c e t h i s t i m e was u s u a l l y s p e n t on o t h e r work. C o m p a r i s o n s a r e b a s e d o n l y on g e n e r a l p a t t e r n s of f e m a l e a p p e a r a n c e and not on d e t a i l e d r e c o r d s of e a c h i n d i v i d u a l c o u r t s h i p . About 60 t o 100 l i m n e t i c f e m a l e s a p p e a r e d on t h e n e s t i n g a r e a d u r i n g t h e o b s e r v a t i o n s . Not a l l male c o u r t s h i p a t t e m p t s were s u c c e s s f u l ( t h a t i s , r e s u l t e d i n f e m a l e n e s t e n t r y ) ; however, s u c c e s s f u l c o u r t s h i p f o l l o w e d a g e n e r a l p a t t e r n . The l i m n e t i c male a p p r o a c h e d a l i m n e t i c f e m a l e and t h e f e m a l e t h e n swam t o t h e male. The male would t h e n t u r n and l e a d t h e f e m a l e t o t h e n e s t i n e i t h e r a d i r e c t or a m e a n d e r i n g p a t h . L i m n e t i c f e m a l e s a p p e a r e d t o f o l l o w b e h i n d or t o t h e s i d e of t h e male. U n s u c c e s s f u l c o u r t s h i p s o c c u r e d e i t h e r when n e i g h b o u r s i n t e r f e r e d i n t h e c o u r t s h i p or t h e f e m a l e b r o k e o f f t h e c o u r t s h i p s e q u e n c e . The l a t t e r o f t e n o c c u r e d when t h e male was a g g r e s s i v e t o w a r d s t h e f e m a l e . U n f o r t u n a t e l y an a c c u r a t e c o u n t of u n s u c c e s s f u l c o u r t s h i p s was not k e p t ; however, t h e r e was no o b v i o u s p a t t e r n as t h e r e was f o r s u c c e s s f u l c o u r t s h i p s . F i v e i n s t a n c e s were o b s e r v e d where b e n t h i c f e m a l e s r e s p o n d e d t o l i m n e t i c male c o u r t s h i p . In a l l c a s e s , t h e 16 c o u r t s h i p broke o f f before the female a r r i v e d at the nest. T y p i c a l l y , the male approached the female s t r a i g h t on or z i g -zagged and the female swam towards the male i n a head-up pos t u r e . The male then turned and i n i t i a t e d the l e a d - f o l l o w sequence. The female then p o s i t i o n e d h e r s e l f near the male. In one case, a male nipped at the female as she .positioned h e r s e l f and the female f l e d . In the remaining four cases, the males nipped the females e a r l y i n the l e a d - f o l l o w sequence. In three of the four i n s t a n c e s , the bre a k - o f f p o i n t was obvious, s i n c e the males turned and at t a c k e d the benthic females and drove them from t h e i r t e r r i t o r i e s . The other benthic female l e f t the t e r r i t o r y a f t e r s e v e r a l nips- but without a chase. The a g g r e s s i v e a t t a c k s by l i m n e t i c males on benthic females may have been due to the i n c o r r e c t p o s i t i o n i n g of the females. In these f i e l d o b s e r v a t i o n s , the only aspect of benthic female p o s i t i o n i n g that I recorded and that o b v i o u s l y d i f f e r e d from l i m n e t i c female p o s i t i o n i n g , was t h e i r tendency to swim towards the d o r s a l region of the males. Nest ing Benthic Males I observed only two sexual c o n t a c t s between benthic males and benthic females. One r e s u l t e d in nest entry, and the other encounter broke o f f a f t e r the female reached the nest. In both cases the male swam d i r e c t l y to the female and lunged at her. The females responded by swimming to the male. When the male 1 7 s t a r t e d the l e a d - f o l l o w sequence the females i n i t i a l l y f ollowed above and s l i g h t l y behind the male. Only "two c o u r t s h i p s i n v o l v i n g l i m n e t i c females and benthic males were observed. In both cases the encounters were the same; the p a i r s both broke o f f before a l e a d - f o l l o w sequence was i n i t i a t e d . The male's approach c o n s i s t e d of swimming s t r a i g h t to the female. The female responded with a head-up d i s p l a y and swam to the male. The females then p o s i t i o n e d themselves next to the male and the p a i r momentarily remained m o t i o n l e s s . The females then l e f t the male and q u i c k l y swam out of the t e r r i t o r i e s . U n l i k e the l i m n e t i c male-benthic female p a i r i n g , the l i m n e t i c females l e f t the benthic male before any overt sign of ag g r e s s i o n from the male. Both l i m n e t i c females appeared to o r i e n t next to the benthic . male and made no e f f o r t to swim towards the d o r s a l region of the male. The motionless behaviour of both benthic males may have been the r e s u l t of the male w a i t i n g f o r the l i m n e t i c females to o r i e n t p r o p e r l y (that i s , i n the f a s h i o n of benthic females). Since the males f a i l e d to i n i t i a t e l e a d i n g behaviour, presumably the females d i d not r e c e i v e an a p p r o p r i a t e s i g n a l and so departed. U n f o r t u n a t e l y , the sample s i z e of observed c o u r t s h i p behaviour i n benthic males i s small so b e h a v i o u r a l p a t t e r n s in c o u r t s h i p are not as apparent as in l i m n e t i c males; however, the l i m i t e d number of o b s e r v a t i o n s on c o u r t i n g benthic males r e f l e c t s the d i f f i c u l t y i n l o c a t i n g them. The few c o u r t i n g benthic males I observed were l o c a t e d i n dense v e g e t a t i o n , t h e i r nests a p p a r e n t l y concealed beneath the v e g e t a t i o n . Darkly 18 c o l o u r e d benthic males were sometimes observed moving amongst v e g e t a t i o n but I c o u l d not determine i f they were i n t h e i r sexual phase. Poor v i s i b i l i t y prevented s e a r c h i n g f o r benthic males i n water greater than one meter deep. Despite t h i s l a c k of ob s e r v a t i o n s , there i s no reason to suspect that benthic males are l e s s s u c c e s s f u l than l i m n e t i c males i n l e a d i n g females of t h e i r own p o p u l a t i o n to the nest. Table 1. Number of vegetat ion males captured in open areas and dense Benthic Ma 1 e s Limn e t i c Ma 1 e s Open Areas 3 18 Dense V e g e t a t i o n 25 2 S e l e c t i v e Trapping Twenty-three p a i r s of minnow t r a p s were p l a c e d e i t h e r in open areas (11) or i n dense v e g e t a t i o n (12). The r e s u l t s are presented i n Table 1. C l e a r l y , b e n t h i c males are more common i n dense v e g e t a t i o n and l i m n e t i c males are more common i n open areas. The d i f f e r e n c e i s s i g n i f i c a n t (G=32.64; p<0.005). T h i s i s c o n s i s t e n t with the o b s e r v a t i o n that nests of benthic males were 19 i n dense v e g e t a t i o n and nests of l i m n e t i c males were i n open areas. Benthic males were commonly c o l l e c t e d i n areas covered with bladderwort ( U t r i c u l a r i a ) . I d e a l l y , to determine n e s t i n g h a b i t a t each male should be observed on h i s t e r r i t o r y and h i s nest c o l l e c t e d . T h i s was impossible f o r benthic males because of the c o n c e a l i n g v e g e t a t i o n over t h e i r nests and t h e i r e l u s i v e behaviour once t h e i r n e s t i n g h a b i t a t was d i s t u r b e d . Under these circumstances, t h i s t r a p p i n g method p r o v i d e s the best o b s e r v a t i o n a l f i e l d estimate of h a b i t a t d i f f e r e n c e s between l i m n e t i c and benthic males duri n g the breeding season. Abrupt s h i f t s from open areas to dense v e g e t a t i o n appeared to be more common than gradual changes between the two n e s t i n g h a b i t a t s . As a r e s u l t , on three occasions I observed l i m n e t i c and benthic males as t e r r i t o r i a l neighbours separated by a ve g e t a t i o n boundary. When l i m n e t i c and benthic t e r r i t o r i e s abut, g r a v i d females of both phenotypes that c r u i s e through these n e s t i n g areas may f r e q u e n t l y encounter males of both phenotypes. T h i s may be important i n determining the frequency of c o u r t s h i p s between l i m n e t i c s and b e n t h i c s . 20 C o n c l u s i o n The f i e l d o b s e r v a t i o n s c l e a r l y d e m o n s t r a t e t h a t m a l e s and f e m a l e s of b o t h p o p u l a t i o n s come i n c o n t a c t d u r i n g t h e b r e e d i n g s e a s o n . None of t h e i n t e r p o p u l a t i o n c o u r t s h i p s o b s e r v e d i n t h e f i e l d r e s u l t e d i n n e s t e n t r y by t h e f e m a l e . L i m n e t i c m a l e s n e s t i n open a r e a s and b e n t h i c males n e s t i n d e n s e v e g e t a t i o n ; however, t h e f r e q u e n c y of i n t e r p h e n o t y p i c p a i r i n g s may i n c r e a s e when l i m n e t i c and b e n t h i c males become t e r r i t o r i a l n e i g h b o u r s . T h i s s u g g e s t s t h e p o s s i b i l i t y t h a t when i n t e r p h e n o t y p i c p a i r i n g s o c c u r , b e h a v i o u r a l d i f f e r e n c e s e a r l y i n c o u r t s h i p a l l o w males and f e m a l e s t o d i s t i n g u i s h i n d i v i d u a l s of t h e i r own p o p u l a t i o n . 21 MATE SELECTION EXPERIMENTS Introduct ion If Enos Lake s t i c k l e b a c k s mate randomly then males and females should not p r e f e r mates of t h e i r own p o p u l a t i o n . To determine i f mating i s random two types of mate s e l e c t i o n experiments were conducted: female c h o i c e (two males and one female), and male c h o i c e (two females and one male). In both experiments the c r i t e r i o n f o r r e c o r d i n g a c h o i c e was female entry i n t o the nest. Mate c h o i c e s were recorded i n a c l o s e d s e q u e n t i a l t e s t design (Cole, 1962) u n t i l e i t h e r s i g n i f i c a n c e or n o n s i g n i f i c a n c e was achieved. The minimum number of t r i a l s necessary f o r h i g h l y s i g n i f i c a n t (p<0.0l)- r e s u l t s i s seven. N o n s i g n i f i c a n t r e s u l t s r e q u i r e acceptance of the n u l l hypothesis ( i n t h i s case random mating). S i g n i f i c a n t r e s u l t s r e q u i r e acceptance of one of two a l t e r n a t i v e hypotheses: mating i s a s s o r t a t i v e , or mating i s d i s a s s o r t a t i v e . Which of these hypotheses i s accepted depends on whether females tend to enter nests of males from t h e i r own ( a s s o r t a t i v e mating), or the other, p o p u l a t i o n ( d i s a s s o r t a t i v e mating). 22 Methods A l l mate s e l e c t i o n experiments were conducted in the l a r g e experimental a q u a r i a . F i s h were introduced through a c l e a r p l e x i g l a s s box f i t t e d with a trap-door and operated by a p u l l -s t r i n g . (a) Male mate s e l e c t i o n (one male, two females): Twenty-four hours before any male mate s e l e c t i o n , a black p a r t i t i o n was p l a c e d between the males. T h i s prevented the males from i n t e r f e r i n g with one another. A l i m n e t i c and benthic female, both p r e v i o u s l y t e s t e d f o r r e c e p t i v i t y , were then p l a c e d in the p l e x i g l a s s box and suspended in the experimental a q u a r i a . A f t e r both females had resumed normal swimming in the p l e x i g l a s s box and had seen the c o u r t i n g male, the s t r i n g was p u l l e d and the females r e l e a s e d . T h i s confinement u s u a l l y l a s t e d from 3 to 5 minutes. The experiment ended when one of the females entered the nest. I f there was no nest entry a f t e r 15 minutes, the females were removed. A d i f f e r e n t p a i r of females were used f o r each male mate s e l e c t i o n t r i a l . Only nest entry was scored as the c r i t e r i o n of c h o i c e . F i v e l i m n e t i c males and four benthic males were used in these t r i a l s as w e l l as fourteen l i m n e t i c females and fourteen benthic females. (b) Female mate s e l e c t i o n (one female, two males): The c l e a r p l e x i g l a s s p a r t i t i o n between two n e s t i n g males (a l i m n e t i c and b e n t h i c ) was removed and the p l e x i g l a s s box suspended in the t e s t tank between the males. F i f t e e n minutes l a t e r , e i t h e r a l i m n e t i c or benthic f e m a l e ( p r e v i o u s l y t e s t e d f o r r e c e p t i v i t y ) 23 was p l a c e d i n the box. Again, t h i s confinement l a s t e d from 3 to 5 minutes. An experiment ended with e i t h e r nest entry or removal of the female a f t e r 15 minutes. Only nest entry was scored. A ~ d i f f e r e n t female was used i n each t r i a l . F i v e d i f f e r e n t p a i r s of males were used f o r the female mate s e l e c t i o n t r i a l s as w e l l as seven l i m n e t i c females and seven benthic females. R e s u l t s Female Mate S e l e c t ion When given a choice of males, females entered the nests of males of t h e i r own p o p u l a t i o n i n 7 out of 7 t r i a l s u s i ng f i v e p a i r s of males. These t r i a l s i n c l u d e d aquarium-length chases, c o u r t s h i p , nest maintenance, and t e r r i t o r i a l d i s p l a y s and f i g h t i n g . D espite the a c t i v i t y , however, the males appeared to spend most of t h e i r time i n t h e i r own h a l f of the aquarium. Apparently, the 15 minute p e r i o d between removing the p a r t i t i o n and i n t r o d u c i n g females was s u f f i c i e n t f o r the males to e s t a b l i s h a boundary that was c l o s e to the p a r t i t i o n ' s o r i g i n a l p o s i t i o n ; however, the 15 minute p e r i o d was s u f f i c i e n t l y short to prevent the more pugnacious benthic males from dominating the e n t i r e aquarium. Male c o u r t s h i p was never c o n f i n e d to one h a l f of the tank. C o u r t s h i p s o f t e n i n v o l v e d a male pursuing the • female i n t o the 24 other h a l f of the tank and t e r r i t o r i a l f i g h t i n g o f t e n r e s u l t e d from these i n c u r s i o n s . C u r i o u s l y , c o u r t s h i p sequences were r a r e l y i n t e r r u p t e d by the neighbouring male; however, in p a i r i n g s between p o p u l a t i o n s , females f r e q u e n t l y f l e d the a g g r e s s i v e a t t a c k s of the male. T h i s a g g r e s s i v e behaviour r e s u l t e d in lengthy chases and ended most of the u n s u c c e s s f u l c o u r t s h i p sequences. Male Mate S e l e c t ion When given a cho.ce of females, males s u c c e s s f u l l y courted females of t h e i r own p o p u l a t i o n (that i s , females entered t h e i r nests) i n 7 out or 7 t r i a l s u s i n g seven d i f f e r e n t p a i r s of females. The only combination that d i d not produce a p o s i t i v e female reponse was benthic males c o u r t i n g l i m n e t i c females. Benthic males f r e q u e n t l y b i t l i m n e t i c females, and a f t e r an i n i t i a l a t t a c k , l i m n e t i c females would remain in a corner or attempt to f l e e . Although l i m n e t i c males b i t benthic females, benthic females d i d show p o s i t i v e responses to l i m n e t i c male approaches. None of the l i m n e t i c male-benthic female p a i r i n g s reached the nest s i n c e they broke-off at some p o i n t d u r i n g the l e a d - f o l l o w sequence. Females were never observed to i n t e r r u p t any b e h a v i o u r a l i n t e r a c t i o n between the male and the other female. 25 Conclusion The mate s e l e c t i o n experiments c l e a r l y demonstrate that mating i s non-random; males and females p r e f e r mates of t h e i r own p o p u l a t i o n . Since i n t e r p h e n o t y p i c p a i r i n g s were u n s u c c e s s f u l , the mate s e l e c t i o n r e s u l t s are c o n s i s t e n t with f i e l d o b s e r v a t i o n s suggesting that b e h a v i o u r a l d i f f e r e n c e s d u r i n g i n t e r p h e n o t y p i c c o u r t s h i p allow males and females to d i s t i n g u i s h i n d i v i d u a l s of t h e i r own population.. In g e n e r a l , the data demonstrate that males and females can d i s t i n g u i s h each' other based on behaviour or morphology, or both. 26 COURTSHIP EXPERIMENTS I n t r o d u c t i o n S t i c k l e b a c k c o u r t s h i p i s regarded as a stimulus-response chain that s t a r t s with the i n i t i a l contact between the sexes and ends with the females e n t e r i n g the nest and d e p o s i t i n g her eggs (Wootton, 1976). The s u c c e s s f u l completion of c o u r t s h i p i s thought to be dependent on the c o r r e c t exchange of s i g n a l s (behaviour and p o s i t i o n i n g ) by both sexes (Tinbergen, 1951). If t h i s i s t r u e , then d i f f e r e n c e s i n c o u r t s h i p behaviour p o t e n t i a l l y provide a b a s i s f o r mate d i s c r i m i n a t i o n between the l i m n e t i c and benthic p o p u l a t i o n s . Based on other s t u d i e s (Semler, 1971), morphological d i f f e r e n c e s probably play a r o l e in phenotypic r e c o g n i t i o n between l i m n e t i c benthic s t i c k l e b a c k s in Enos Lake; however, the problem of simultaneously manipulating form and c o l o u r e l i m i n a t e d t e s t i n g m o rphological d i f f e r e n c e s as c r i t e r i a f o r m a i n t a i n i n g r e p r o d u c t i v e i s o l a t i o n . U n t i l r e c e n t l y (McPhail and Hay, i n p r e s s ) , b e h a v i o u r a l and p o s i t i o n a l d i f f e r e n c e s i n c o u r t s h i p were not recorded in s t u d i e s of a s s o r t a t i v e . mating between p a r a p a t r i c p o p u l a t i o n s of G. a c u l e a t u s (McPhail, 1969; Moodie, 1972a; Hay and McPhail, 1975). McPhail and Hay ( i n press) found s u f f i c i e n t d i f f e r e n c e s in male c o u r t s h i p , p a r t i c u l a r l y i n the i n i t i a l approach to females, t o provide a b a s i s f o r p o s i t i v e a s s o r t a t i v e mating between p a r a p a t r i c freshwater and marine p o p u l a t i o n s . In Enos Lake, the r e s u l t s from f i e l d and mate s e l e c t i o n o b s e r v a t i o n s 27 i n d i c a t e t h a t d i f f e r e n c e s i n c o u r t s h i p may c o n t r i b u t e t o b e h a v i o u r a l i s o l a t i o n b e t w een l i m n e t i c s a nd b e n t h i c s . The p u r p o s e o f t h i s s e c t i o n i s t o e x a m i n e male c o u r t s h i p a nd f e m a l e r e s p o n s e f o r 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 ween t h e p o p u l a t i o n s . M e t h o d s A s i n g l e r e c e p t i v e l i m n e t i c o r b e n t h i c f e m a l e was p l a c e d i n a b l a c k e n e d j a r s i t u a t e d i n an a q u a r i u m c o r n e r away f r o m t h e m a l e ' s n e s t . A f t e r 5 m i n u t e s t h e l i d was l i f t e d a n d t h e f e m a l e e v e n t u a l l y swam o u t . R e c o r d i n g s t a r t e d when t h e f e m a l e l e f t t h e j a r . T h i s was d e s i g n a t e d t h e b e g i n n i n g o f a c o u r t s h i p t r i a l . F e m a l e s were u s e d when a v a i l a b l e a n d i n t r o d u c e d i n a s e q u e n c e w i t h o u t o b v i o u s b i a s t o p r e v e n t c o n d i t i o n i n g t h e m ale t o a s e t p a t t e r n o f f e m a l e i n t r o d u c t i o n s . The b l a c k e n e d j a r s were l e f t i n t h e a q u a r i u m s t o h a b i t u a t e t h e m a l e s t o t h e i r p r e s e n c e . A l l c o u r t s h i p e x p e r i m e n t s were c o n d u c t e d i n t h e l a r g e e x p e r i m e n t a l a q u a r i a . N i n e l i m n e t i c m a l e s , t h i r t e e n l i m n e t i c f e m a l e s , f i v e b e n t h i c m a l e s , and t w e l v e b e n t h i c f e m a l e s were u s e d i n t h e c o u r t s h i p t r i a l s . S i n c e i n some c o u r t s h i p t r i a l s t h e f e m a l e d i d n o t r e s p o n d , t h e f o l l o w i n g c r i t e r i a were u s e d t o d e t e r m i n e w h e t h e r o r n o t a c o u r t s h i p t r i a l was i n c l u d e d i n a n a l y s i s : ( 1 ) t h e f e m a l e must have shown a p o s i t i v e r e s p o n s e t o t h e male w i t h i n 3 m i n u t e s o f t h e s t a r t o f t h e t r i a l , a n d ( 2 ) t h e f e m a l e must have r e a c h e d t h e n e s t w i t h i n 15 m i n u t e s . I t was my e x p e r i e n c e i n o b s e r v i n g 28 s t i c k l e b a c k c o u r t s h i p t h a t i f a f e m a l e d i d not r e s p o n d t o a male or r e a c h h i s n e s t w i t h i n t h e s p e c i f i e d t i m e l i m i t s t h e f e m a l e was not r e a d y t o mate. In t h e few c a s e s where t h i s o c c u r r e d , I p r o b a b l y m i s i n t e r p r e t e d t h e f e m a l e ' s i n i t i a l r e s p o n s e t o t h e t e s t male. T a b l e 2 summarizes t h e number of c o u r t s h i p t r i a l s t h a t r e s u l t e d i n n e s t e n t r y by t h e f e m a l e . T a b l e 2. Number of c o u r t s h i p t r i a l s e n d i n g w i t h n e s t e n t r y by th e f e m a l e . L i m n e t i c L i m n e t i c B e n t h i c Male + Male + Male + L i m n e t i c B e n t h i c B e n t h i c Female Female Female N e s t 1 0 •3 8 E n t r y No N e s t 5 5 7 E n t r y I f t h e s e c r i t e r i a were s a t i s i f i e d t h e t r i a l was u s e d i n a n a l y s i s . A t r i a l ended when t h e f e m a l e e i t h e r e n t e r e d t h e n e s t , or one of t h e f o l l o w i n g e v e n t s o c c u r r e d : (1) a f t e r a "show n e s t e n t r a n c e " b o u t , t h e male b i t t h e f e m a l e 3 or more t i m e s and t h e a t t a c k l a s t e d 5 o r more s e c o n d s ; (2) a f t e r a "show n e s t e n t r a n c e " b o u t , t h e f e m a l e d i d not p o s i t i v e l y r e s p o n d w i t h i n 3 m i n u t e s ; (3) 30 m i n u t e s had e l a s p e d s i n c e t h e s t a r t of r e c o r d i n g . The b i t i n g and t i m e c r i t e r i a were a r b i t r a r i l y c h o s e n end p o i n t s , however, s i n c e i n t h e f i e l d male a g g r e s s i o n or f e m a l e r e t i c e n c e n e v e r r e s u l t e d i n n e s t e n t r y t h e s e c r i t e r i a a p p e a r r e a s o n a b l e . In a l l c o u r t s h i p t r i a l s , t h e t i m e i n t e r v a l 29 for r e c o r d i n g was 0.5 seconds and 19 behaviours were recorded (Table 3). Table 3. Behaviours recorded in c o u r t s h i p experiments 1. Male d i r e c t approach to female. 2. Male z i g - z a g approach to female. 3. Male b i t e female. 4. Female swim to male. 5. Male lead female i n d i r e c t path. 6. Male lead female i n meandering path. 7. Female follow above male. 8. Female follow beside male. 9. Female f o l l o w behind male. 10. Male d o r s a l p r i c k female. 11. Male show nest entrance to female: 12. Female below male and f a c i n g nest entrance. 13. Female not below male or f a c i n g nest entrance. 14. Female poke snout i n t o nest entrance. 15. Female swim away from nest and male. 16. Female enter nest. 17. Male perform nest maintenance behaviours (fanning, g l u i n g , pushing, e t c . ) . 18. Male creep through nest. 19. N u l l (male swim i n any d i r e c t i o n or remain mot i o n l e s s ) . 30 R e s u l t s The f o l l o w i n g number of c o u r t s h i p t r i a l s were i n c l u d e d i n data a n a l y s i s : (1) f i f t e e n t r i a l s with l i m n e t i c males and females; (2) f i f t e e n t r i a l s with benthic males and females, and (3) e i g h t t r i a l s with l i m n e t i c males and benthic females. Because benthic males tend to be h i g h l y a g g r e s s i v e towards l i m n e t i c females, the p a i r i n g of benthic males and l i m n e t i c females never r e s u l t e d i n s u c c e s s f u l c o u r t s h i p . Such p a i r i n g s were c h a r a c t e r i z e d by intense p u r s u i t and b i t i n g by the male. In extreme cases, the male ended up attempting to swallow the female. A f t e r f i v e attempts i n v o l v i n g three d i f f e r e n t benthic males, i t was c l e a r , at l e a s t under l a b o r a t o r y c o n d i t i o n s , that t h i s combination was not going to succeed. In these attempts, as in the male mate s e l e c t i o n experiments, l i m n e t i c females were never observed to show a p o s i t i v e response to benthic males. Since t h i s combination (benthic males and l i m n e t i c females) produced no p o s i t i v e responses, the t r i a l s were excluded from the s t a t i s t i c a l a n a l y s i s ; however, the f i e l d o b s e r v a t i o n s and a few examples from the female mate s e l e c t i o n experiments i n d i c a t e that a p o s i t i v e response by a l i m n e t i c female to a c o u r t i n g benthic male i s p o s s i b l e . The lack of p o s i t i v e responses in the c o u r t s h i p experiments were probably due to the f o r c e d nature of the encounter and the c o n f i n e d space of the arena. In a l l other .combinations of males and females, c o u r t s h i p u s u a l l y produced p o s i t i v e responses. For convenience in a n a l y s i s , c o u r t s h i p behaviour was d i v i d e d i n t o three phases: ( l ) male approach and 31 female response; (2) male lead and female f o l l o w , and (3) male and female together at the nest. At any time d u r i n g the f i r s t two phases the male might leave the female and swim alone to h i s nest to conduct nest maintenance a c t i v i t i e s . Since the end of c o u r t s h i p was determined by the f i s h and not the observer, a female that i n i t i a l l y r e f u s e d to enter the nest c o u l d be approached by the c o u r t i n g male s e v e r a l times. T h i s behaviour was a l s o observed in the f i e l d . In the l a b o r a t o r y , van den Assem (1967) r e f e r r e d to the male's behaviour of approaching the female a f t e r t h e i r i n i t i a l nest v i s i t as " c o u r t s h i p c y c l e s " . " C o u r t s h i p c y c l e s " occur when a male induces a female to swim to the nest with him more than once. Otherwise, a c o u r t s h i p ended e i t h e r with the female in the nest or when a male a g g r e s s i v e l y pursued a female f o r more than f i v e seconds. The f i e l d o b s e r v a t i o n s suggest that when the l a t t e r occurs a female would normally leave the t e r r i t o r y . Table 4 l i s t s the number of c o u r t s h i p c y c l e s for a l l combinations of l i m n e t i c s and b e n t h i c s as w e l l as the p r o b a b i l i t y of a female e n t e r i n g the nest on her f i r s t nest v i s i t . Most nest e n t r i e s o c c u r r e d i n the f i r s t " c o u r t s h i p c y c l e " with few in l a t e r nest v i s i t s . The data i n d i c a t e that a s u b s t a n t i a l part of the behaviour used in c o u r t s h i p a n a l y s i s came from the i n i t i a l encounter and f i r s t " c o u r t s h i p c y c l e " . 32 T a b l e 4. The number of c o u r t s h i p c y c l e s and t h e p r o b a b i l i t y of t h e f e m a l e e n t e r i n g t h e n e s t on t h e f i r s t c y c l e . L i m n e t i c L i m n e t i c B e n t h i c M a l e + Male + Male + L i m n e t i c B e n t h i c B e n t h i c Female Female Female C y c l e T o t a l : 15 8 1 5 One I n : 9 4 10 P r o b . of e n t r y : 0.6 0.5 0.67 C y c l e T o t a l : 4 4 1 Two I n : 0 0 0 C y c l e T o t a l : 4 2 1 T h r e e I n : 1 1 0 C y c l e T o t a l : 3 0 0 F o u r I n : 0 0 0 M a l e A p p r o a c h and Female Response T a b l e 5 summarizes t h e male a p p r o a c h and f e m a l e r e s p o n s e d a t a . I t i n c l u d e s t h e number o f p o s i t i v e f e m a l e r e s p o n s e s , t h e number of n e g a t i v e f e m a l e r e s p o n s e s , and t h e m a l e ' s d e s t i n a t i o n a f t e r a n e g a t i v e f e m a l e r e s p o n s e (by d e f i n i t i o n , a l l p o s i t i v e r e s p o n s e s r e s u l t i n t h e male moving t o w a r d s h i s n e s t ) . B e n t h i c male a g g r e s s i o n t o w a r d s l i m n e t i c f e m a l e s p r e v e n t e d t h e r e c o r d i n g of c o u r t s h i p r e s p o n s e s between t h i s c o m b i n a t i o n ; however, t h e r e a r e q u a l i t a t i v e d i f f e r e n c e s between t h i s and o t h e r male and f e m a l e c o m b i n a t i o n s . T h e s e d i f f e r e n c e s w i l l be i n d i c a t e d when a p p r o p r i a t e . S i n c e t h e i n i t i a l s t a g e s of c o u r t s h i p a r e l i k e l y t o be i m p o r t a n t i n s i g n a l l i n g whether a s t i c k l e b a c k i s l i m n e t i c or 33 T a b l e 5. Summary of male a p p r o a c h and f e m a l e r e s p o n s e . The p o s i t i v e r e s p o n s e t o a m a l e ' s a p p r o a c h i s " f e m a l e swim t o male". The n e g a t i v e f e m a l e r e s p o n s e t o a male's r e s u l t s i n "male swim a l o n e t o n e s t " . P e r c e n t of t o t a l f e m a l e • r e s p o n s e i n e a c h c a t e g o r y i s i n p a r e n t h e s e s . L i m n e t i c Male + L i m n e t i c Female L i m n e t i c Male + B e n t h i c Female B e n t h i c M a l e + B e n t h i c F e m ale Female Swim t o M a l e ; P o s i t i v e 1 03 (92.8) 58 (78.4) 69 (78.4) M a l e Swim A l o n e t o N e s t ; Negat i v e 8 (7.2) 1 6 (21.6) 19 (21.6) b e n t h i c , t h e y a r e a l s o p r o b a b l y i m p o r t a n t i n m a i n t a i n i n g r e p r o d u c t i v e i s o l a t i o n . To d e t e r m i n e i f t h e r e - w e r e d i f f e r e n c e s i n t h e i n i t i a l s t a g e s o f c o u r t s h i p , male a p p r o a c h and f e m a l e r e s p o n s e was compared i n terms o f : (1) t h e number and t y p e of male a p p r o a c h e s ( d i r e c t , z i g - z a g , o r b i t e ) p e r m i n u t e ; (2) t h e r e l a t i o n s h i p between t h e t y p e o f male a p p r o a c h and f e m a l e r e s p o n s e , and (3) t h e d u r a t i o n of male a p p r o a c h and p o s i t i v e f e m a l e r e s p o n s e . L i m n e t i c males b i t b e n t h i c f e m a l e s more f r e q u e n t l y t h a n t h e y b i t l i m n e t i c f e m a l e s ( T a b l e 6 ) . L i m n e t i c m a l e s n e v e r c h a s e d e i t h e r l i m n e t i c or b e n t h i c f e m a l e s t o t h e e x c l u s i o n of a l l o t h e r b e h a v i o u r . Such p e r s i s t e n t a g g r e s s i o n o n l y o c c u r r e d when b e n t h i c m a l e s were combined w i t h l i m n e t i c f e m a l e s . B e n t h i c m a l e s p e r s i s t e n t l y b i t and c h a s e d l i m n e t i c f e m a l e s but n e v e r d e m o n s t r a t e d t h i s l e v e l of a g g r e s s i o n t o w a r d s b e n t h i c f e m a l e s . T a b l e 7.summarizes p o s i t i v e f e m a l e r e s p o n s e s t o t h e t h r e e 34 Table 6. Mean number and standard error of bites per minute, zig-zag bouts per minute, and female swim to male bouts per minute. The test s t a t i s t i c i s the Wilcoxon-Mann-Whitney U (two-tailed; Sokal and Rohlf, 1981). Data presented in Appendixes 1 and 2. Limnet ic Male + Limnet ic Female Limnet ic Male + Benthic Female Benthic Male + Benthic Female Bites per Minute 1 . 1 7±0.35 2.75±0.66 3.38±0.94 Zig-zag Bouts per Minute 1 .47±0.39 0.68±0.24 0.66±0.24 Female Swim to 1 .91±0.28 1.55±0.31 1 .73±0.25 Male Bouts per Minute Bites per Minute Z ig-zags Bouts per Minute Female Swim to Male'Bouts per Minute Limnetic Male + Limnetic Female vs Benthic Male + Benthic Female U=165.5 p<0.005 U=1 53 p<0. 1 U-1 27 p>0. 1 Limnetic Male + Limnetic Female vs Limnetic Male + Benthic Female U=95 p<0.025 U=76.5 p>0. 1 U=71 p>0. 1 Benthic Male + Benthic Female vs Limnetic Male + Benthic Female U=63 p>0. 1 U=65 p>0. 1 U=65.5 p>0. 1 types of male approach. The data demonstrate that limnetic male zig-zags receive more positive responses from both limnetic and benthic females than the other forms of approach; however, benthic male bites receive a positive response from benthic females (G=37.54; p<0.005) more often than limnetic male bites. 35 Table 7. Summary of positive female responses to the three types of male approach. G values are from a 3X2 contingency table with the Williams correction factor (Sokal and Rohlf, 1981). Limnet ic Male + Limnet ic Female Limnet ic Male + Benthic Female Benthic Male + Benthic Female Straight Approach 1 7 20 1 7 Zig-Zag Approach 55 26 7 Bite Approach 31 1 2 45 Limnet ic Benthic I Male Male + Limnetic Female vs + Benthic Female G= 37 .54; p<0.005 Limnet ic Limnet ic Male Male + Limnetic vs + Benthic Female Female G= 6. 70; p<0.05 Benthic : Limnet ic Male + Benthic Female vs Male + Benthic Female G= 30 .76; p<0.005 When approached by limnetic males, there i s a difference in the d i s t r i b u t i o n of positive responses by limnetic and benthic females to the three types of limnetic male approach (G=6.70; p<0.05). Table 7 indicates that benthic females respond most frequently to bites from benthic males and to zig-zags from limnetic males. This difference in benthic female response to the two types of male approach is s i g n i f i c a n t (G=30.76; p<0.005). Limnetic females never showed a posit i v e response to benthic male approaches. Since the only intensity difference in male approach is the 36 b i t i n g f r e q u e n c y of b e n t h i c males ( T a b l e 6 ) , t h e l i m n e t i c f e m a l e s may r e s p o n d more p o s i t i v e l y t o t h e l e s s a g g r e s s i v e a p p r o a c h ( z i g z a g ) and b e n t h i c f e m a l e s more p o s i t i v e l y t o t h e most f r e q u e n t a p p r o a c h ( b i t e ) . The a s s u m p t i o n u n d e r l y i n g s u c h a c o n c l u s i o n i s t h a t male z i g z a g g i n g and b i t i n g a r e t h e same f o r l i m n e t i c s and b e n t h i c s . T h i s s i m i l a r i t y i s assumed i n some s t u d i e s ( W i l z , 1973); however, t h e r e i s a g r a d i e n t i n i n t e n s i t y and p e r s i s t e n c e f o r e a c h c a t e g o r y of a p p r o a c h b e h a v i o u r . F o r example, " b i t i n g " c a n g r a d e from g e n t l e n i p s or b u t t s and l u n g e s t h r o u g h t o v i o l e n t a t t a c k s . The s h o r t d u r a t i o n of t h e s e e v e n t s p r e c l u d e s r e l i a b l e i d e n t i f i c a t i o n and r e c o r d i n g under any c a t e g o r y e x c e p t a g e n e r a l " b i t i n g " . C o m p a r i s o n s between l i m n e t i c s and b e n t h i c s d e m o n s t r a t e t h a t male z i g z a g and b i t e a p p r o a c h e s a r e c l e a r l y d i f f e r e n t ; b e n t h i c f e m a l e s r e s p o n d more t o t h e z i g z a g of l i m n e t i c males t h a n t h e z i g z a g s o f b e n t h i c m a l e s , and l i m n e t i c f e m a l e s o c c a s i o n a l l y r e s p o n d t o t h e b i t e s of l i m n e t i c m ales b ut were n e v e r o b s e r v e d t o r e s p o n d t o b i t e s from b e n t h i c m a l e s i n c o u r t s h i p e x p e r i m e n t s ( T a b l e 7 ) . F i g u r e s 2, 3, and 4 a r e c u m u l a t i v e f r e q u e n c i e s of t h e d u r a t i o n s of male a p p r o a c h and p o s i t i v e f e m a l e r e s p o n s e ( d a t a i n A p p e n d i x 3 ) . No d i s t i n c t i o n i s made between t h e t y p e s of male a p p r o a c h . Based on t h e K o l m o g o r o v - S m i r n o v t w o - t a i l e d t e s t , t h e r e i s a s i g n i f i c a n t d i f f e r e n c e between t h e c u m u l a t i v e f r e q u e n c y d i s t r i b u t i o n i n f i g u r e s 2 and 4. They d e m o n s t r a t e t h a t l i m n e t i c male a p p r o a c h e s and p o s i t i v e b e n t h i c f e m a l e r e s p o n s e s t a k e s i g n i f i c a n t l y l o n g e r t h a n t h e same b e h a v i o u r s i n c o m p a r i s o n s between t h e two k i n d s o f males ( F i g u r e s 2 and 3) and between 37 p o p u l a t i o n s ( F i g u r e 4 ) . However, t h e d i f f e r e n c e i n F i g u r e 3 i s n o t s i g n i f i c a n t a t t h e 0.05 l e v e l . Whether t h i s d e l a y i s due t o male or f e m a l e r e t i c e n c e c a n n o t be d e t e r m i n e d ; however, t h e f i g u r e s s u g g e s t t h a t when l i m n e t i c m ales c o u r t b e n t h i c f e m a l e s , one or b o t h of t h e s e x e s h e s i t a t e s i n t h e i n i t i a l s t a g e s o f c o u r t s h i p . Thus, even t h o u g h t h e i n c o m p a t i b i l i t y between l i m n e t i c m ales and b e n t h i c f e m a l e s i s not as p r o n o u n c e d as t h a t between b e n t h i c males and l i m n e t i c f e m a l e s , t h e p a t t e r n a p p e a r s t o be t h e same, and d i f f e r e n c e s e a r l y i n t h e c o u r t s h i p sequence may h e l p t o m a i n t a i n r e p r o d u c t i v e i s o l a t i o n between t h e f o r m s . Under u n c o n s t r a i n e d f i e l d c o n d i t i o n s , t h e l o n g e r d u r a t i o n of l i m n e t i c male a p p r o a c h and b e n t h i c f e m a l e r e s p o n s e may l e a d t o a b r e a k i n c o u r t s h i p . M a l e L e a d and Female F o l l o w The l e a d - f o l l o w s e q u e n c e i s a s e r i e s of b e h a v i o u r s where t h e male l e a d s t h e f e m a l e from t h e e n c o u n t e r p o i n t t o h i s n e s t . T h i s phase of c o u r t s h i p b e g i n s a f t e r t h e f e m a l e r e s p o n d s p o s i t i v e l y t o t h e male's a p p r o a c h . I t s t a r t s when t h e male t u r n s away from t h e f e m a l e and t h e f e m a l e assumes a f o l l o w i n g p o s i t i o n n e a r t h e male. The number of p o s i t i v e (male and f e m a l e a t n e s t ) and n e g a t i v e (male and f e m a l e b r e a k o f f ) l e a d - f o l l o w s e q u e n c e s , as w e l l as male d e s t i n a t i o n i n c a s e s where t h e c o u r t s h i p b r o k e -o f f , a r e l i s t e d i n T a b l e 8. At t h e b e g i n n i n g of ...a l e a d - f o l l o w s e q u e n c e t h e f e m a l e can 38 Figure 2. Cumulative frequency d i s t r i b u t i o n s of the durations of male approach and p o s i t i v e female response for limnetic males courting limnetic and benthic females. Right pointing triangles represent data from limnetic males courting limnetic females. Crosses represent data from limnetic males courting benthic females (Kolmogorov-Smirnov test, two-tailed; p < 0 . 0 0 1 ) . 0. 5- 10/ 15. EO- E5. 30 DURATION (HALF-SECONDS) gure 3. Cumulative frequency d i s t r i b u t i o n s of the d u r a t i o n s of male approach and p o s i t i v e female response f o r l i m n e t i c and benthic males c o u r t i n g benthic females. Downward p o i n t i n g t r i a n g l e s represent data from be n t h i c males c o u r t i n g benthic females. Crosses represent data from l i m n e t i c males c o u r t i n g benthic females (Kolmogorov-Smirnov t e s t , t w o - t a i l e d ; p<0 . 1 ) . 41 l-C" 0- 5. 10. 15- SO- E5- 30-DURATION (HALF-SECONDS) 42 F i g u r e 4. C u m u l a t i v e f r e q u e n c y d i s t r i b u t i o n s of t h e d u r a t i o n s o f male a p p r o a c h and p o s i t i v e f e m a l e r e s p o n s e f o r m a l e s c o u r t i n g f e m a l e s from t h e i r own p o p u l a t i o n . R i g h t p o i n t i n g t r i a n g l e s r e p r e s e n t d a t a from l i m n e t i c m a les c o u r t i n g l i m n e t i c f e m a l e s . Downward p o i n t i n g t r i a n g l e s r e p r e s e n t d a t a from b e n t h i c m a l e s c o u r t i n g b e n t h i c f e m a l e s ( K o l m o g o r o v - S m i r n o v t e s t , t w o - t a i l e d ; p<0.025) . 43 1'CL DURATION (HALF-SECONDS) 44 T a b l e 8. Summary of t h e number of p o s i t i v e (male and f e m a l e a t t h e n e s t ) and n e g a t i v e (male and f e m a l e b r e a k - o f f ) l e a d - f o l l o w s e q u e n c e s . L i m n e t i c M a l e + L i m n e t i c Female L i m n e t i c M a le + B e n t h i c Female B e n t h i c M a l e + B e n t h i c Female Male and Female a t N e s t ( p o s i t i v e ) 63 1 3 18 M a l e Swim A l o n e t o N e s t ( n e g a t i v e ) 22 8 1 2 M a l e new a p p r o a c h t o Female ( n e g a t i v e ) 10 21 . 2 8 assume one o f t h r e e p o s i t i o n s r e l a t i v e t o t h e male: (1) e i t h e r above t h e male; (2) or a l o n g s i d e t h e male, or (3) b e h i n d t h e male. S i n c e t h e r e l a t i v e p o s i t i o n o f t h e male and f e m a l e c a n change d u r i n g a l e a d - f o l l o w s e q u e n c e , i t i s l i k e l y t h a t f e m a l e p o s i t i o n a t t h e b e g i n n i n g of a s e q u e n c e i s more i m p o r t a n t t h a n p o s i t i o n d u r i n g a s e q u e n c e . The d a t a p r e s e n t e d i n T a b l e 9 show c l e a r d i f f e r e n c e s between l i m n e t i c and b e n t h i c f e m a l e s i n t h e i r p o s i t i o n a t t h e s t a r t of a l e a d - f o l l o w s e q u e n c e . L i m n e t i c f e m a l e s p o s i t i o n t h e m s e l v e s a l o n g s i d e t h e male more f r e q u e n t l y t h a n t h e y p o s i t i o n t h e m s e l v e s above t h e male, whereas b e n t h i c f e m a l e s u s u a l l y p o s i t i o n t h e m s e l v e s above t h e male (G=62.04; p<0.005). When p a r t n e r e d w i t h l i m n e t i c m a l e s , l i m n e t i c f e m a l e s s t a r t e d more f r e q u e n t l y f r o m b e h i n d t h e male, whereas w i t h t h e same males. 45 T a b l e 9. Female p o s i t i o n r e l a t i v e t o t h e male a t t h e s t a r t of a l e a d - f o l l o w s e q u e n c e . G - t e s t w i t h W i l l i a m s c o r r e c t i o n f a c t o r b a s e d on 3X2 c o n t i n g e n c y t a b l e ( S o k a l and R o h l f , 1981). L i m n e t i c Male + L i m n e t i c Female L i m n e t i c M a l e + B e n t h i c Female B e n t h i c Male + B e n t h i c Female Above M a l e 3 8 35 A l o n g s i d e M a l e 36 23 5 B e h i n d M a l e 52 1 1 . 26 L i m n e t i c M a l e + L i m n e t i c Female vs B e n t h i c M a l e + B e n t h i c Female G= 62 .04; p<0. 005 L i m n e t i c M a l e + L i m n e t i c vs L i m n e t i c M a l e + B e n t h i c Female Female G= 1 5 .21; p<0. 005 B e n t h i c M a l e + B e n t h i c Female v s L i m n e t i c M a l e + B e n t h i c Female G= 31 .08; p<0. 005 b e n t h i c f e m a l e s t e n d e d t o s t a r t a l o n g s i d e t h e male (G=15.21; p<0.005). However, when b e n t h i c f e m a l e s were p a r t n e r e d w i t h l i m n e t i c m ales t h e i r s t a r t i n g p o s i t i o n was d i f f i c u l t t o d e t e r m i n e . T h i s was b e c a u s e t h e y were l a r g e r t h a n t h e l i m n e t i c m a l e s , and t h u s o f t e n a p p e a r e d t o be above as w e l l as b e h i n d t h e more t e r e t e l i m n e t i c m ale. As a r e s u l t , ^ f e m a l e s t a r t i n g p o s i t i o n was not as d i s t i n c t a s i n t h e l i m n e t i c m a l e - l i m n e t i c f e m a l e c o m b i n a t i o n . B e n t h i c f e m a l e s p o s i t i o n e d t h e m s e l v e s a l o n g s i d e l i m n e t i c m a l e s more f r e q u e n t l y t h a n above t h e male, whereas t h e y p o s i t i o n t h e m s e l v e s above b e n t h i c m a l e s more f r e q u e n t l y t h a n 46 a l o n g s i d e t h e male (G=31.08; p<0.005). T h i s d i f f e r e n c e may s i m p l y r e f l e c t my u n c e r t a i n t y c o n c e r n i n g t h e p o s i t i o n of b e n t h i c f e m a l e s r e l a t i v e t o l i m n e t i c m a l e s . T a b l e 10. The number of d i r e c t o r m e a n d e r i n g male l e a d s a t t h e s t a r t of l e a d - f o l l o w s e q u e n c e s . G - t e s t w i t h W i l l i a m s c o r r e c t i o n f a c t o r i s b a s e d on 2X2 c o n t i n g e n c y t a b l e ( S o k a l and R o h l f , 1981) . L i m n e t i c L i m n e t i c M a l e + Mal e + L i m n e t i c B e n t h i c Female Female B e n t h i c M a l e +. B e n t h i c F e m ale D i r e c t L e a d 62 14 1 1 Meander i n g L e a d 34 28 55 L i m n e t i c Male + L i m n e t i c Female vs B e n t h i c M a l e + B e n t h i c Female G= 38 .35; p<0.005 L i m n e t i c Male + L i m n e t i c Female v s L i m n e t i c M a l e + B e n t h i c Female G= 1 1 .46; p<0.005 B e n t h i c M a l e + B e n t h i c Female v s L i m n e t i c M a l e + B e n t h i c Female G= 3. 84; p>0.1 C i r c l e l e a d i n g ( t h a t i s , m e a n d e r i n g ) and d o r s a l p r i c k i n g by t h e male s e r v e a s s i g n a l s t o t h e f e m a l e t o s t o p f o l l o w i n g and r e m a i n s t a t i o n a r y w h i l e t h e male r e t u r n s a l o n e t o t h e n e s t (Wootton, 1976). These b e h a v i o u r s o c c u r when t h e male i s i n an a g g r e s s i v e s t a t e ( W i l z , 1970a,b). In Enos L a k e , d o r s a l p r i c k i n g was r a r e l y o b s e r v e d e i t h e r i n l i m n e t i c s or b e n t h i c s . I n s t e a d , 47 m e a n d e r i n g l e a d s by b o t h k i n d s of m a l e s were common. When c o u r t i n g l i m n e t i c f e m a l e s , l i m n e t i c m a l e s t e n d t o use a d i r e c t l e a d t o t h e n e s t more f r e q u e n t l y t h a n a m e a n d e r i n g l e a d . In c o n t r a s t , b e n t h i c m a l e s c o u r t i n g b e n t h i c f e m a l e s t e n d t o use a m e a n d e r i n g l e a d more f r e q u e n t l y t h a n a d i r e c t l e a d ( T a b l e 10; G=38.35; p<0.005). However, when c o u r t i n g b e n t h i c f e m a l e s , l i m n e t i c m ales use a m e a n d e r i n g l e a d more o f t e n t h a n a d i r e c t l e a d (G=11.46; p<0.005). S i n c e b o t h l i m n e t i c and b e n t h i c m a les t e n d t o use a m e a n d e r i n g l e a d when f o l l o w e d by a b e n t h i c f e m a l e , p e r h a p s b e n t h i c f e m a l e s p r o v i d e a cue t o m ales t h a t d e t e r m i n e s t h a t l e a d i n g b e h a v i o u r w i l l be m e a n d e r i n g . C e r t a i n l y t h e g e n e r a l body shape of t h e f e m a l e s i s d i f f e r e n t and c o u l d s u p p l y a c u e . In a d d i t i o n , d i f f e r e n c e s i n f e m a l e p o s i t i o n a t t h e s t a r t of a l e a d - f o l l o w s e quence a r e p r o b a b l y a l s o i n v o l v e d i n d e t e r m i n i n g male b e h a v i o u r a t t h i s phase i n c o u r t s h i p . The f i e l d o b s e r v a t i o n s s u p p o r t t h i s h y p o t h e s i s . In t h e f i e l d , most of t h e o b s e r v e d c o u r t s h i p s between forms ended i n t h e e a r l y s t a g e s of t h e l e a d - f o l l o w s e q u e n c e . T a b l e 11 summarizes t h e d a t a on male d e s t i n a t i o n when a l e a d - f o l l o w s e q u e n c e b r e a k s - o f f b e f o r e r e a c h i n g t h e n e s t . When c o u r t i n g f e m a l e s of t h e i r own p o p u l a t i o n , l i m n e t i c males r e t u r n t o t h e i r n e s t more o f t e n t h a n t h e y a t t e m p t t o s t a r t a new a p p r o a c h t o t h e f e m a l e , whereas b e n t h i c m a l e s a t t e m p t a new a p p r o a c h t o t h e f e m a l e more f r e q u e n t l y t h a n r e t u r n t o t h e i r n e s t (G=10.74; p<0.005). A p p a r e n t l y t h i s p a t t e r n depends on t h e p h e n o t y p e of t h e f e m a l e , s i n c e l i m n e t i c m a l e s when c o u r t i n g b e n t h i c f e m a l e s r e v e r s e t h e i r b e h a v i o u r and show more new 48 Table 11. Male d i s t i n a t i o n when the male and female break-away from l e a d - f o l l o w sequences. G-test with W i l l i a m s c o r r e c t i o n f a c t o r i s based on 2X2 contingency t a b l e (Sokal and Rohlf, 1981) . Limnetic Limnetic Male + Male + Limnetic Benthic Female Female Benthic Male + Benthic Female New Approach 10 21 to Female 28 Swim to Nest 22 8 1 2 Limnetic Male + Limnetic Female vs Benthic Male + Benthic Female G= 1 0 .74; p<0.005 Limnetic Male + Limnetic Female vs Limnetic Male + Benthic Female G= 1 0 .38; p<0.005 Benthic Male + Benthic Female vs Limnetic Male + Benthic Female G= 0. 05; p>0.1 approaches to females than r e t u r n s to the nest (G=10.38; p<0.005). Thus, when c o u r t i n g benthic females, there i s no d i f f e r e n c e between benthic and l i m n e t i c males in t h e i r response to an u n s u c c e s s f u l l e a d - f o l l o w sequence (G=0.05; p>0.1). Males sometimes b i t females d u r i n g negative l e a d - f o l l o w sequences (Table 12), but s i n c e males r a r e l y b i t females during p o s i t i v e l e a d - f o l l o w sequences, these data were not i n c l u d e d in the a n a l y s i s . I was not able to determine i f these b i t e s by l i m n e t i c and benthic males d i f f e r e d , or i f they d i f f e r e d from the b i t e s that o c c u r r e d during the male's approach. When c o u r t i n g females of t h e i r own p o p u l a t i o n , l i m n e t i c males tended 49 Table 12. The number of u n s u c c e s s f u l l e a d - f o l l o w sequences i n c o r p o r a t i n g male b i t e s versus sequences without male b i t e s . G-test with W i l l i a m s c o r r e c t i o n f a c t o r i s based on 2X2 contingency t a b l e (Sokal and Roh l f , 1981). Limnetic L i m n e t i c Male + Male + Limnetic Benthic Female Female Benthic Male + Benthic Female B i t e s 7 4 21 No B i t e s 25 25 19 Li m n e t i c Male + Limnetic Female vs Benthic Male + Benthic Female G = 7. 09; p<0.0l Li m n e t i c Male + Limnetic Female vs L i m n e t i c Male + Benthic Female G = 0. 65; p>0.1 Benthic Male + Benthic Female vs L i m n e t i c Male + Benthic Female G = 1 1 .45; p<0.005 to b i t e l e s s than benthic males (G= 7.09; p<0.0l between p o p u l a t i o n s , and G=11.45; p<0.005 between males) d u r i n g the l e a d - f o l l o w sequence. For l i m n e t i c males, t h i s d i f f e r e n c e i n b i t i n g d u r i n g a\ l e a d - f o l l o w sequence was independent of female phenotype (G=0.65; p>0.1). Male and Female at the Nest F o l l o w i n g a s u c c e s s f u l l e a d - f o l l o w sequence, the male and female a r r i v e at the nest and go through a s e r i e s of behaviours 50 where the male shows the female the nest entrance. T h i s phase begins when both sexes reach the nest. The male shows the nest entrance by p o i n t i n g h i s snout i n t o the entrance and r a p i d l y opens and c l o s e s h i s mouth. Apparently the female uses the male's behaviour and p o s i t i o n as a guide and p o s i t i o n s h e r s e l f beneath the male. The phase ends e i t h e r with nest entrance or the departure of the female from the nest area. When a male was showing h i s nest entrance to a female, I c l a s s i f i e d the female's p o s i t i o n as e i t h e r below the male and f a c i n g the nest entrance or not below the male. The l a t t e r c ategory takes i n t o account the many p o s i t i o n s a female can assume in the nest area r e l a t i v e to the male. For the purposes of t h i s study, .female p o s i t i o n below the male and f a c i n g the nest entrance was an e s s e n t i a l p r e r e q u i s i t e to the female t r y i n g the nest entrance. Females t r y the nest entrance by poking t h e i r snouts i n t o the entrance. Nest entry i s achieved when the female c o n t i n u e s to push at the entrance and e v e n t u a l l y wiggles i n t o the nest. If a female does not enter she may stay i n the nest area or leave the nest zone. The nest zone i s the area around the nest d e f i n e d by the p i e p l a t e on which the nest i s b u i l t . The male may stop showing the entrance before the female has entered. T h i s u s u a l l y r e s u l t s in the male and female remaining motionless w i t h i n the nest zone. At t h i s time the male u s u a l l y t r i e s to p o s i t i o n himself above the female. The data comparing the mean frequency of females in or out of p o s i t i o n ( r e l a t i v e to the male) are presented i n Table 13. The f r e q u e n c i e s are expressed as the number of times a behaviour 51 T a b l e 13. Mean f r e q u e n c y and s t a n d a r d e r r o r p e r c o u r t s h i p c y c l e of f e m a l e s i n or out o f p o s i t i o n r e l a t i v e t o t h e male ( s e e t e x t f o r d e s c r i p t i o n ) . The t e s t s t a t i s t i c i s t h e W i l c o x o n - M a n n - W h i t n e y U ( t w o - t a i l e d ; S o k a l and R o h l f , 1981). D a t a p r e s e n t e d i n A p p e n d i x 4. L i m n e t i c L i m n e t i c M a l e + Male + L i m n e t i c B e n t h i c Female Female B e n t h i c Male + B e n t h i c Female In P o s i t i o n 3.38±1.91 2.96±1.01 3.0±0.88 Out of P o s i t i o n . 1.29±0.99 2.44±0.75 4.09±1.26 L i m n e t i c M a l e + L i m n e t i c Female In vs B e n t h i c Male + B e n t h i c Female Out p o s i t i o n ; U=125.5; p>0.1 of p o s i t i o n ; U=194; p<0.005 L i m n e t i c M a l e + L i m n e t i c Female In vs L i m n e t i c M a l e + B e n t h i c Female Out p o s i t i o n ; U=75; p>0.1 of p o s i t i o n ; U=96; p<0.025 B e n t h i c M a l e + B e n t h i c Female In vs L i m n e t i c M a l e + B e n t h i c Female Out p o s i t i o n ; U=69.5; p>0.1 of p o s i t i o n ; U=65.5; p>0.1 was o b s e r v e d p e r c o u r t s h i p c y c l e . W i t h m a l e s of t h e i r own p h e n o t y p e , b e n t h i c f e m a l e s were out o f p o s i t i o n more f r e q u e n t l y p e r c o u r t s h i p c y c l e t h a n l i m n e t i c f e m a l e s (U=194; p<0.005). T h i s i s a l s o t r u e when l i m n e t i c males and p a r t n e r e d w i t h b e n t h i c f e m a l e s (U=96; p<0.025). F o r b e n t h i c f e m a l e s , r e m a i n i n g out of p o s i t i o n was i n d e p e n d e n t of t h e male form (U=65.5; p > 0 . l ) . T h e r e was no d i f f e r e n c e i n t h e number of t i m e s f e m a l e s t r i e d t h e n e s t e n t r a n c e ( T a b l e 14). The l e n g t h of t i m e between a r r i v a l a t t h e n e s t and t h e f e m a l e t r y i n g t h e n e s t e n t r a n c e was measured d u r i n g t h e f i r s t 52 Table 14. Mean frequency and standard e r r o r per c o u r t s h i p c y c l e of females t r y i n g the nest entrance. The t e s t s t a t i s t i c i s the Wilcoxon-Mann-Whitney U ( t w o - t a i l e d ; Sokal and Rohlf, 1981). Data presented i n Appendix 4. Limn e t i c L i m n e t i c Male + Male + Limnetic Benthic Female Female Benthic Male + Benthic Female Females Try Nest 2.8111.45 1.44±0.32 Entrance 1.8010.49 Limne t i c Male + Limnetic Female vs Benthic Male + Benthic Female U= 1 1 3 .5; p>0.1 Limn e t i c Male + Limnetic Female vs L i m n e t i c Male + Benthic Female U= 66; p>0.1 Benthic Male + Benthic Female vs Limnetic Male + Benthic Female U= 65. 5; p>0.1 c o u r t s h i p c y c l e of a l l t r i a l s (Table 15). T h i s data p r o v i d e s a means of comparing the amount of time i t takes f o r a female to t r y the nest entrance. The data suggest that females may d i s t i n g u i s h between the nests of l i m n e t i c and benthic males. C e r t a i n l y , benthic females took longer than l i m n e t i c females to t r y the nest entrance when courted by l i m n e t i c males (U=103; P<0.01). In the f i e l d , none of the observed c o u r t s h i p s between l i m n e t i c s and b e n t h i c s reached the nest. Thus, i t i s not c l e a r i f male and female behaviours at the nest are c r i t i c a l f o r mate d i s c r i m i n a t i o n ; however, they are p o t e n t i a l l y important s i n c e the female can i n s p e c t the nest, and based on c o n s t r u c t i o n or 53 T a b l e 15. Mean l e n g t h o f time ( h a l f s e c o n d s ) and s t a n d a r d e r r o r between a r r i v a l a t t h e n e s t and t h e f e m a l e t r y i n g t h e n e s t e n t r a n c e . The t e s t s t a t i s t i c i s t h e W i l c o x o n - M a n n - W h i t n e y U ( t w o - t a i l e d ; S o k a l and R o h l f , 1981). D a t a p r e s e n t e d i n A p p e n d i x 5. L i m n e t i c L i m n e t i c B e n t h i c Male + Male + Male + L i m n e t i c B e n t h i c B e n t h i c Female Female Female L e n g t h of Time between Female A r r i v a l 11.4±5.22 5 6 . 7 5 ± 1 9 . 2 5 36.8±19.01 and T r y N e s t E n t r a n c e L i m n e t i c M a l e + L i m n e t i c Female v s B e n t h i c M a l e + B e n t h i c Female U= 155.5; p<0.1 L i m n e t i c M a l e + L i m n e t i c Female v s L i m n e t i c M a l e + B e n t h i c Female U= 103; p<0.0l B e n t h i c M a l e + B e n t h i c Female v s L i m n e t i c M a l e + B e n t h i c Female U= 8 5.5; p<0.1 c o n d i t i o n , d e t e r m i n e i f t h e n e s t i s s u i t a b l e . S i n c e b e n t h i c f e m a l e s t a k e l o n g e r t h a n l i m n e t i c f e m a l e s t o t r y t h e n e s t e n t r a n c e o f l i m n e t i c m a l e s , i t i s p o s s i b l e t h a t l i m n e t i c n e s t s may d i f f e r from b e n t h i c n e s t s . A l t h o u g h no a t t e m p t was made t o document d i f f e r e n c e s i n n e s t s , i t i s l i k e l y t h a t t h e n e s t s of t h e two t y p e s a r e d i f f e r e n t , s i n c e t h e y a r e made from m a t e r i a l s f o u n d i n d i f f e r e n t m i c r o - h a b i t a t s and a r e c o n s t r u c t e d by m a l e s of d i f f e r e n t m o r p h o l o g y . 54 Male Swim t o t h e N e s t A l o n e In my e x p e r i m e n t a l d e s i g n , when a male l e a v e s t h e f e m a l e a f t e r e i t h e r an a p p r o a c h s e q u e n c e o r a l e a d - f o l l o w s e q u e n c e he swims a l o n e t o h i s n e s t . A s e q u e n c e ends when t h e male r e a c h e s h i s n e s t . The d a t a on t h i s b e h a v i o u r a r e summarized i n T a b l e 16. When c o u r t i n g t h e i r own f e m a l e s , l i m n e t i c males l e a v e t h e f e m a l e and v i s i t t h e n e s t more f r e q u e n t l y a f t e r a l e a d - f o l l o w s e q uence t h a n a f t e r an a p p r o a c h s e q u e n c e , whereas b e n t h i c males l e a v e t h e i r f e m a l e s more f r e q u e n t l y a f t e r an a p p r o a c h sequence (G=5.24; p<0.025). F o r l i m n e t i c m a l e s t h i s d i f f e r e n c e a p p e a r s t o depend on f e m a l e p h e n o t y p e . L i m n e t i c males most f r e q u e n t l y l e a v e b e n t h i c f e m a l e s a f t e r an a p p r o a c h sequence whereas t h e y most f r e q u e n t l y l e a v e l i m n e t i c f e m a l e s a f t e r a l e a d - f o l l o w s e q u e n c e (G=6.39; p<0.025). The r e a s o n f o r t h i s d i f f e r e n c e i s n o t c l e a r , i t may r e f l e c t an a g g r e s s i v e r e s p o n s e by t h e m a l e s t o w a r d s l a r g e f e m a l e s . C o n c l u s i o n The c o u r t s h i p e x p e r i m e n t s c l e a r l y d e m o n s t r a t e t h a t t h e r e a r 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 between l i m n e t i c and b e n t h i c s t i c k l e b a c k s . W i t h f e m a l e s of t h e i r own p o p u l a t i o n , b e n t h i c m a l e s a r e more a g g r e s s i v e i n t h e i r a p p r o a c h and l e a d i n g s e q u e n c e s t h a n l i m n e t i c m a l e s . In t h e b e g i n n i n g of t h e l e a d -55 Table 16. The number of times males l e f t females f o r t h e i r nests a f t e r approach or l e a d - f o l l o w sequences. G-test with W i l l i a m s c o r r e c t i o n f a c t o r i s based on 2X2 contingency t a b l e s (Sokal and Rohlf, 1981). Limnetic L i m n e t i c Male + Male + Limnetic Benthic Female Female Benthic Male + Benthic Female Male Leave female A f t e r an Approach 8 16 1 9 Male Leave Female A f t e r a Lead-Follow 18 8 1 2 Limnetic Male Benthic Male + Limnetic Female vs + Benthic Female G= 5. 24; p<0.025 Limnetic Male Limnetic Male + Limnetic Female vs + Benthic Female G= 6. 39; p<0.025 Benthic Male + Benthic Female vs Limnetic Male + Benthic Female G= 0. 16; p>0. 1 f o l l o w phase, benthic females tend to p o s i t i o n themselves above benthic males whereas l i m n e t i c females tend to p o s i t i o n themselves a l o n g s i d e l i m n e t i c males. Male c o u r t s h i p behaviour i s sometimes dependent on the phenotype of the female courted; benthic males b i t and chased l i m n e t i c females whereas l i m n e t i c males b i t and l e d benthic females i n a meandering path to the nest. Since the mate choice experiments i n d i c a t e d t o t a l p o s i t i v e a s s o r t a t i v e mating between l i m n e t i c s and b e n t h i c s , i t i s l i k e l y that the b e h a v i o u r a l d i f f e r e n c e s documented i n t h i s s e c t i o n c o n t r i b u t e to r e p r o d u c t i v e i s o l a t i o n between the l i m n e t i c and benthic sticklebacks in Enos Lake. 57 DISCUSSION The r e s u l t s suggest t h a t : (1) d e s p i t e d i f f e r e n c e s i n n e s t i n g h a b i t a t , c o u r t s h i p between l i m n e t i c s and b e n t h i c s occurs in nature, but (2) that such c o u r t s h i p s appear to break o f f because of b e h a v i o u r a l d i f f e r e n c e s i n c o u r t s h i p , and (3) given a c h o i c e , males and females of each p o p u l a t i o n p r e f e r mates of t h e i r own p o p u l a t i o n . In a d d i t i o n , there are d i f f e r e n c e s i n c o u r t s h i p behaviour between l i m n e t i c s and b e n t h i c s and these d i f f e r e n c e s are g r e a t e s t i n the e a r l y stages of c o u r t s h i p . These r e s u l t s g e n e r a l l y support p r e v i o u s c o n c l u s i o n s that the l i m n e t i c and benthic p o p u l a t i o n s of Gasterosteus i n Enos Lake c o n s t i t u t e s i b l i n g s p e c i e s ( W i t h l e r , 1980; McPhail, i n p r e p ) . For sympatric, s i b l i n g species,, c o u r t s h i p should s i g n a l the i n d i v i d u a l ' s species a f f i l i a t i o n and thus s u s t a i n mate d i s c r i m i n a t i o n (Mayr, 1963). L i l e y (1966) p r e d i c t e d that in such cases s e l e c t i o n w i l l favour b e h a v i o u r a l d i f f e r e n c e s e a r l y i n c o u r t s h i p s i n c e t h i s p r o v i d e s the most economical means of mate d i s c r i m i n a t i o n . He emphasized that the e a r l y sequences of c o u r t s h i p behaviour show g r e a t e r s p e c i e s - s p e c i f i c i t y than the s i g n a l s and responses which occur l a t e r i n the sequence. If h y b r i d s are s e l e c t e d a g a i n s t in Enos Lake, then mate d i s c r i m i n a t i o n between the l i m n e t i c and benthic p o p u l a t i o n s should occur e a r l y in c o u r t s h i p . My r e s u l t s c l e a r l y demonstrate that i n Enos Lake s t i c k l e b a c k s the most pronounced d i f f e r e n c e s occur e a r l y in c o u r t s h i p (that i s , male approach, i n i t i a l female response, and the beginning of the l e a d - f o l l o w phase). The longer d u r a t i o n of male approach and female response when 58 l i m n e t i c males were p a i r e d with benthic females suggests that from the time the i n d i v i d u a l s become aware of one another to the a c t u a l encounter poi n t both sexes are a s s e s s i n g one another. If males and females are a s s e s s i n g each other during t h i s time, t h i s may be when morphological d i f f e r e n c e s determine i f c o u r t s h i p w i l l proceed. The c o n f i n e d nature of the aquarium probably encouraged p r o p o r t i o n a t e l y more i n t e r p h e n o t y p i c p a i r i n g s to reach the nest than would normally occur in the f i e l d ; however, c o u r t s h i p between l i m n e t i c s and b e n t h i c s does occur i n the f i e l d so morphological d i f f e r e n c e s are. not always s u f f i c i e n t i n p r e v e n t i n g c o u r t s h i p from porceeding. C e r t a i n l y , once c o u r t s h i p proceeds b e h a v i o u r a l d i f f e r e n c e s are important i n s i g n a l l i n g s p e c i e s a f f i l i a t i o n . F i e l d o b s e r v a t i o n s from Enos Lake i n d i c a t e that d i f f e r e n c e s in the beginning of the l e a d - f o l l o w phase may be an important " b r e a k - o f f " p o i n t f o r p a i r i n g s between forms. Laboratory r e s u l t s c o n f i r m the d i f f e r e n c e i n female p o s i t i o n . Limnetic females tend to p o s i t i o n themselves a l o n g s i d e l i m n e t i c males and benthic females tend to p o s i t i o n themselves above benthic males. Because they are l a r g e r , benthic females i n e v i t a b l y adopt an i n c o r r e c t p o s i t i o n r e l a t i v e to l i m n e t i c males. My data i n d i c a t e that males of both forms tend to i n i t i a l l y l e a d benthic females in a meandering path. T h i s d i f f e r e n c e suggests that benthic females may cause an a g g r e s s i v e s t a t e i n c o u r t i n g males; however, d u r i n g l e a d - f o l l o w sequences, benthic males b i t e benthic females more f r e q u e n t l y than l i m n e t i c males and t h i s a g g r e s s i v e r e l a t i o n s h i p does not e x i s t between l i m n e t i c males and l i m n e t i c females. 59 Limnetic males tend to i n i t i a l l y l e ad l i m n e t i c females i n a d i r e c t path to the nest. Reasons for an a g g r e s s i v e s t a t e towards benthic females are a matter of s p e c u l a t i o n , but i n many s p e c i e s of f i s h , l a r g e females appear to e l i c i t more aggression from c o u r t i n g males than small females (Barlow, 1968; Weber and Weber, 1976). In s i m i l a r s t u d i e s , d i f f e r e n c e s i n the l e a d - f o l l o w sequence duri n g mating t e s t s between P u n g i t i u s p u n g i t i u s and G. a c u l e a t u s (Wilz, 1970c) and between G. wheatlandi and G. a c u l e a t u s (Mclnerny, 1969) were found to be important i n m a i n t a i n i n g r e p r o d u c t i v e i s o l a t i o n . Previous work on mate s e l e c t i o n i n Gasterosteus suggests that mate ch o i c e between separate forms in parapatry i s based on morphology and male c o l o u r . The r o l e of n u p t i a l c o l o u r i n mate ch o i c e i n Gasterosteus was demonstrated by Semler (1971). He used a p o p u l a t i o n polymorphic for male t h r o a t c o l o u r and showed that females p r e f e r e n t i a l l y enter nests of r e d - t h r o a t e d males and that the red t h r o a t was the c r i t e r i o n of c h o i c e . In a study u s i n g anadromous and r e s i d e n t p o p u l a t i o n s of t h r e e s p i n e s t i c k l e b a c k , Hay and McPhail (1975) demonstrated that i n c h o i c e s i t u a t i o n s males and females were most l i k e l y to mate with i n d i v i d u a l s from t h e i r own p o p u l a t i o n . Moodie's (1972a) c h o i c e experiments i n v o l v e d s t i c k l e b a c k s from Mayer Lake and i n d i c a t e d a degree of i s o l a t i o n between l i m n e t i c and stream d w e l l i n g s t i c k l e b a c k s that appears s i m i l a r to that i n Enos Lake. In Mayer Lake, however, the l i m n e t i c and stream forms are p a r a p a t r i c and the males d i f f e r i n n u p t i a l c o l o u r . In the C h e h a l i s R i v e r system, McPhail (1969) showed that females from a l l o p a t r i c red 60 and black p o p u l a t i o n s p r e f e r e n t i a l l y o r i e n t to c o u r t i n g red males. T h i s preference was not as pronounced i n black females from a black p o p u l a t i o n that abutted a g a i n s t a red p o p u l a t i o n . In c o - e x i s t i n g s u n f i s h (Lepomi s) f i e l d evidence demonstrates: (1) that a d u l t s s e t t l e onto t e r r i t o r i e s at approximately the same time; (2) there are s u b t l e d i f f e r e n c e s i n n e s t i n g h a b i t a t ; (3) the s p e c i e s are f r e q u e n t l y t e r r i t o r i a l neighbours, and (4) there are r e l a t i v e l y few h y b r i d s (Keenleyside, 1978). These d i f f e r e n c e s are a l s o c h a r a c t e r i s t i c of Enos Lake l i m n e t i c and benthic s t i c k l e b a c k s n e s t i n g i n the f i e l d ; apparent d i f f e r e n c e s i n n e s t i n g h a b i t a t , t e r r i t o r i a l c o ntact between l i m n e t i c s and b e n t h i c s , and r e l a t i v e l y few hyb r i d s (<5% of e l e c t r o p h o r e t i c samples, W i t h l e r , 1980; pers . comm.). Under these circumstances, and based on the evidence i n t h i s study and i n McPhail and Hay (1982), d i f f e r e n c e s in the e a r l y stages of c o u r t s h i p behaviour between l i m n e t i c and benthic s t i c k l e b a c k s are important f o r m a i n t a i n i n g r e p r o d u c t i v e i s o l a t i o n . However, i t i s not yet apparent what s e l e c t i v e disadvantage i s a s s o c i a t e d with being a h y b r i d i n Enos Lake. For s u n f i s h and c i c h l i d s p e c i e s n e s t i n g together, d i f f e r e n c e s i n the e a r l y stages of c o u r t s h i p behaviour are important when e c o l o g i c a l and seasonal d i f f e r e n c e s in breeding are i n s u f f i c i e n t f o r m a i n t a i n i n g r e p r o d u c t i v e i s o l a t i o n . Using three s p e c i e s of s u n f i s h ( b l u e g i l l , Lepomi s macrochirus; pumpkinseed, L. gibbosus; longear, L. . m e g a l o t i s ) , Keenleyside (1967) found that n e s t i n g males p r e f e r r e d to court t h e i r own 61 females and that males can recognize females d u r i n g the e a r l y stages of c o u r t s h i p . S t e e l e and Keenleyside (1971) completed the mate s e l e c t i o n experiments and demonstrated that females p r e f e r e n t i a l l y o r i e n t towards c o n s p e c i f i c males. The c o n c l u s i o n from these two s t u d i e s was that mate d i s c r i m i n a t i o n and b e h a v i o u r a l d i f f e r e n c e s during the e a r l y stages of c o u r t s h i p were major components c o n t r i b u t i n g to r e p r o d u c t i v e i s o l a t i o n between sympatric p o p u l a t i o n s . In c h o i c e experiments, B a y l i s (1976a) demonstrated complete i s o l a t i o n between sympatric p o p u l a t i o n s of the midas c i c h l i d (Cichlasoma c i t r i n e l l u m ) and the arrow c i c h l i d (C. zaliosum) c o - e x i s t i n g i n Lake Apoyo, Nicaragua. U n f o r t u n a t e l y , f i e l d evidence i s incomplete- f o r the two s p e c i e s but i t appears that there i s broad o v e r l a p i n breeding seasons and a d u l t s are thought to be t e r r i t o r i a l neighbours. He demonstrated that the g r e a t e s t d i f f e r e n c e i n c o u r t s h i p between the two c i c h l i d s p e c i e s occurred in the e a r l y stages of c o u r t s h i p sequences ( B a y l i s , 1976b). I n t e r e s t i n g l y , B a y l i s (1976b) found that both c i c h l i d s p e c i e s performed the same c o u r t s h i p d i s p l a y s but i n d i f f e r e n t f r e q u e n c i e s . T h i s i s the case f o r male approach behaviour i n Enos Lake l i m n e t i c s and b e n t h i c s . Changes in the frequency of i n i t i a l c o u r t s h i p behaviours appear to be the most economical means of s i g n a l l i n g s p e c i e s a f f i l i a t i o n between sympatric s p e c i e s n e s t i n g in c l o s e p r o x i m i t y . Mate d i s c r i m i n a t i o n by both sexes i s an important i s o l a t i n g mechanism in sympatric f i s h s p e c i e s , and d i f f e r e n c e s , p a r t i c u l a r l y in the e a r l y stages of c o u r t s h i p , appear to 62 i d e n t i f y c o n s p e c i f i c and h e t e r o s p e c i f i c mates. In other animals the a v a i l a b l e evidence supports t h i s g e n e r a l i t y . For example, a c o u s t i c s i g n a l l i n g i n sympatric w o l f " s p i d e r s (Schizocosa sp., S t r a t t o n and Uetz, 1981); a d v e r t i s i n g c a l l s i n Western Grebe co l o u r forms (Aechmophorus o c c i d e n t a l i s , N u e c h t e r l e i n , 1981), and a g g r e s s i v e b i t i n g in the f i r s t stages of c o u r t s h i p between chromosome races of mole r a t s (Spalax sp., Nevo, 1969). N a t u r a l s e l e c t i o n i s thought to shape the r u l e s determining behaviour sequences so that the r e p r o d u c t i v e output of an i n d i v i d u a l i s optimized i n an environment to which i t i s adapted ( S i b l y and McFarland, 1976). There may be d i f f e r e n c e s i n the r u l e s determining the outcome of c o u r t s h i p s sequences in Enos Lake l i m n e t i c and benthic s t i c k l e b a c k s . For example, a f t e r l e a v i n g the female during a l e a d - f o l l o w sequence, males can go to the nest or reapproach the female. With females from t h e i r own p o p u l a t i o n , l i m n e t i c males most f r e q u e n t l y r e t u r n to the nest, whereas benthic males reapproach the female. Since l i m n e t i c males a l s o reapproach benthic females, male response appears to depend on female phenotype. Male behaviour p r i o r to r e t u r n i n g to the nest r e f l e c t s t h i s p a t t e r n of dependence on female phenotype. When c o u r t i n g benthic females, l i m n e t i c and benthic males more f r e q u e n t l y r e t u r n to the nest a f t e r approaching. With l i m n e t i c females, l i m n e t i c males r e t u r n a f t e r a l e a d - f o l l o w sequence. Since approaches to benthic females p r i o r to nest r e t u r n i n c o r p o r a t e mostly b i t i n g , i t appears that the l a r g e r benthic females ( l a r g e r r e l a t i v e to l i m n e t i c females) e l i c i t more agg r e s s i v e responses, at l e a s t from l i m n e t i c males. 63 I t i s not c l e a r i f the a g g r e s s i o n shown by the two types of males i s the same. T h e i r b i t e s and zigzags seem d i f f e r e n t and benthic males t y p i c a l l y b i t e more than l i m n e t i c males, p a r t i c u a r l y when partnered with l i m n e t i c females. In any case, males appear to be choosing behaviour based on female phenotype; benthic males choose to b i t e and chase l i m n e t i c females whereas l i m n e t i c males choose to adopt approach and l e a d i n g behaviours s i m i l a r to those of benthic males when c o u r t i n g benthic females. 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M.Sc. thesis, University of B r i t i s h Columbia, 136p. Wootton, R.J. 1976. The Biology of the Sticklebacks. Academic Press, London. 387p. APPENDIX 1. DURATION (MINUTES) OF COURTSHIP TRIALS. Limnetic Benthic Limnetic Male + Male + Male + T r i a l Limnetic Benthic Benthic Number Female Female Female 1 3.57 3.79 5.47 2 2.15 0.9 4.04 3 2.23 0.67 4.78 4 2.14 1 .47 7.35 5 1 .07 0.55 0.93 6 26. 1 2 3.03 4.59 7 0.2 1 .23 9.1 1 8 1.15 7.75 0.83 9 1 .58 0.93 10 1.08 1 .53 1 1 5.36 2.63 1 2 2.73 0.33 13 9.02 9.03 1 4 13.73 12.02 1 5 0.73 4.26 70 APPENDIX 2. MEAN FREQUENCY PER COURTSHIP TRIAL OF ZIG-ZAG BOUTS PER MINUTE, BITES PER MINUTE, AND FEMALE SWIM TO MALE BOUTS PER MINUTE. Limnetic Male + Limnet ic Female Benthic Male + Benthic Female Limnet ic Male + Benthic Female T r i a l Number 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 Z i g -zag Bite Fm. Swim 0.28 0.93 0 0.93 2.80 0.50 5.0 3.48 1 .90 0.93 2.80 0 0.33 2.11 0 1.96 0.84 1.40 1.86 1.35 2.24 0 0 0 2.37 2.80 1.87 0.69 0.80 5.0 5.0 0.87 0.87 2.53 1 .85 1.31 2.24 0.37 1.83 0 0.44 0.36 2.48 1.37 1.37 Z i g -zag Bite Fm. Swim 3.64 0.26 1.32 0.53 1.11 14.44 1.11 2.99 2.99 1.49 0.68 1.36 2.04 1 .82 0 0.66 3.30 0.66 0 3.25 2.44 0.13 3.74 1.55 2.1.5 1.08 1.08 0 1.96 3.27 0 4.18 0.76 0 0 0.11 1 .22 0.89 0 4.33 1.75 0 3.03 Z i g -zag Bite Fm. Swim 0 3.11 2.19 1.24 3.47 3.22 1.26 2.30 1.88 0.27 0.27 0.41 0 2.15 1.08 1.09 6.54 1.53 . 1.54 2.96 0.88 0 1.20 1.20 7.51 1.64 71 APPENDIX 3. DURATION (HALF-SECONDS) OF MALE APPROACH AND POSITIVE FEMALE RESPONSE (FEMALE SWIM TO MALE). D u r a t i o n i n h a l f -s e c o n d s L i m n e t i c Male + L i m n e t i c Female B e n t h i c Male + B e n t h i c Female L i m n e t i c M a l e + B e n t h i c Female Cum. Cum. F r e q . F r e q . P r o b . Cum. Cum. F r e q . F r e q . P r o b . Cum. Cum. F r e q . F r e q . P r o b 1 1 1 0.01 0 0 0 0 0 0 2 4 5 0.05 1 1 0.01 1 1 0.02 3 1 7 22 0.21 9 10 0.14 2 3 0.06 4 15 37 0.35 6 16 0.23 3 6 0.11 5 19 56 0.53 5 21 0.30 4 10 0.19 6 1 0 66 0.63 1 2 33 0.47 7 1 7 0.32 7 5 71 0.68 8 41 0.59 2 19 . 0.36 8 9 80 0.76 5 46 0.66 5 24 0.45 9 5 85 0.81 4 50 0.71 5 29 0.55 10 4 89 0.85 2 52 0.74 3 32 0.60 1 1 1 90 0.86 3 55 0.79 4 36 0.68 1 2 0 90 0.86 3 58 0.83 0 36 0.68 1 3 1 91 0.87 3 61 0.87 . 2 38 0.72 1 4 2 93 0.89 0 61 0.87 0 38 0.72 1 5 '1 94 0.90 . 1 62 0.89 0 38 0.72 16 1 95 0.90 0 62 0.89 2 40 0.75 1 7 1 96 0.91 1 63 0.90 1 41 0.77 18 2 98 0.93 0 63 0.90 1 42 0.79 19 2 100 0.95 0 63 0.90 2 44 0.83 20 1 101 0.96 0 63 0.90 1 4 5 0.85 21 1 1 02 0.97 0 63 0.90 2 47 0.87 22 1 1 03 0.98 1 64 0.91 2 49 0.92 23 0 1 03 0.98 0 64 0.91 0 49 0.92 24 0 1 03 0.98 0 64 0.91 1 50 0.94 25 0 103 0.98 1 65 0.93 1 51 0.96 26 0 1 03 0.98 0 65 0.93 0 51 0.96 27 0 1 03 0.98 0 65 0.93 0 51 0.96 28 0 1 03 0.98 0 65 0.93 1 52 0.98 29 0 103 0.98 0 65 0.93 0 52 0.98 30 0 1 03 0.98 0 65 0.93 0 52 0.98 >30 2 1 05 1 .0 5 70 1.0 1 53 1 .0 72 APPENDIX 4. MEAN FREQUENCY OF FEMALE IN POSITION AT NEST, OUT OF POSITION AT NEST, AND TRYING THE NEST ENTRANCE PER COURTSHIP CYCLE FOR COURTSHIP TRIALS T r i a l Limnet i c Male + Limnet i c Female Benthic Male + Benthic Female Limnet i c Male + Benthic Female Number In Out Try In Out Try In Out Try 1 3.0 0 2.0 6.0 14.0 2.0 1 .0 0 1 .0 2 2.0 1 .0 3.0 1 .0 1 .0 1 .0 1 .0 0.5 0.5 3 1 .0 0 1 .0 1 .0 2.0 1 .0 2.33 2.0 2.33 4 1 .0 0 1 .0 4.0 4.0 4.0 1 .33 4.0 0.67 5 1 .0 0 1 .0 2.0 2.0 2.0 8.0 2.0 3.0 6 3.6 0.6 3.0 13.0 6.0 1 .0 1 .0 1 .0 1 .0 7 1 .0 0 1.0 1 .0 1 .0 1 .0 2.0 3.0 1 .0 8 1 .0 0 1 .0 1.0 1 .0 1 .0 7.0 7.0 2.0 9 1 .0 0 1 .0 1 .0 1 .0 1 .0 1 0 1 .0 0 1.0 1 .0 1 .0 1 .0 1 1 0.64 1.18 0.64 4.0 12.0 1 .0 1 2 1 .33 0.33 1 .0 1 .0 1 .0 1 .0 1 3 30.0 15.0 23.0 7.0 13.33 8.0 1 4 2.15 0.26 1 .53 1 .0 1 .0 1 .0 1 5 1 .0 1 .0 1 .0 1 .0 1 .0 1 .0 73 APPENDIX 5 ARRIVAL AT TIME THE NEST (HALF SECONDS) FROM THE UNTIL THE FEMALE FIRST ENTRANCE FEMALE'S FIRST TRIES THE NEST T r i a l Number L i m n e t i c Male + L i m n e t i c Female B e n t h i c Male + B e n t h i c Female L i m n e t i c Male + B e n t h i c Female 1 5 257 9 2 6 6 65 3 7 34 92 4 4 1 0 167 5 2 5 4 6 5 21 1 2 7 3 1 6 43 8 3 5 62 9 4 4 1 0 5 5 1 1 80 1 69 1 2 3 5 1 3 4 7 1 4 31 3 15 9 5 

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