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Eggs in the nests of males and their effect on mate choice in the three-spined stickleback
Anita. Behav., 1989, 38, 859-865
Eggs in the nests of males and their effect on mate choice in the three-spined stickleback I A N G. J A M 1 E S O N & P A T R I C K W. C O L G A N
Department of Biology, Queen's University, Kingston, Ontario, K7L 3N6, Canada
Abstract. It is thought that female three-spined sticklebacks, Gasterosteus aculeatus, prefer to mate with males whose nests already contain eggs. This is based on the results from a study where gravid females were tested sequentially, first with a male without eggs, and then with a male with eggs. However, that experimental design fails to detect important effects due to male-male competition. Simultaneous and sequential choice experiments were conducted to determine the relative importance of eggs in a nest with respect to female mate choice. In the simultaneous choice tests, males with eggs in their nest were more likely to obtain a second spawning than males without eggs. However, there was little evidence of females actively 'choosing' mates on the basis of presence or absence of eggs. Rather, males that had recently spawned courted more intensely and were more successful at leading females to their nests. In only a few cases could a male's failure to spawn be attributed to a female aborting entry into a nest without eggs. In the sequential choice tests, significantly more females entered and spawned than aborted entry into nests without eggs. Furthermore, aborted entries were not seen exclusively in nests without eggs. The results indicated that the presence of eggs had a general priming effect on courtship behaviour of males, but evidence for active female choice was lacking. Overall, females did not exhibit the behavioural patterns predicted from the hypothesis that females should spawn preferentially in nests with eggs.
The importance of male traits with respect to female mate choice has recently received a great deal of attention (e.g. Bateson 1983). Female preferences for particular traits can result in variation in male mating success. However, it has been notoriously difficult to distinguish the effects of active female choice from those of passive choice in intrasexual competition (Halliday 1983). In fishes with exclusively paternal care, several possible adaptive advantages have been suggested for females choosing males who are already mated (see Rohwer 1978; Ridley & Rechten 1981). There is now evidence in four species that females prefer to spawn in nests that already contain eggs. In three of these species, the tessellated darter, Etheostoma olmstedi (Constanz 1985), the river bullhead, Cottus gobio (Marconato & Bisazza 1986), and the fathead minnow, Pimephales promelas (Unger & Sargent 1988), males defend rocks and crevices, but show little or no courtship behaviour toward gravid females. Females inspect suitable spawning sites defended by the males and nests that already contain eggs receive significantly more new eggs than nests with no eggs. By contrast, male three-spined sticklebacks, Gasterosteus aculeatus, construct a substrate nest out of. vegetation and perform elaborate courtship 0003-3472/89/110859+ 07 $03.00/0
behaviour in order to lead gravid females to their nests. Ridley & Rechten (1981) conducted a sequential choice experiment in which females were presented to single males in isolation. Some females were more likely to enter a nest and spawn than refuse mating if the nest of the male already contained eggs; a refusal took the form of a female putting her snout into the nest and then backing out and swimming away. This study has often been cited as evidence that female sticklebacks prefer to spawn in nests with eggs (e.g. Sargent 1982; Halliday 1983; Fitzgerald & Wootton 1986; Vickcry et al. 1988), yet such a conclusion is difficult to draw from Ridley & Rechten's results. Their initial experiment found that the presence of eggs in a nest did not significantly increase the female's probability of spawning (Ridley & Rechten 1981). In a second experiment they reported that some of the females who had refused to spawn in a nest of a male without eggs were more likely to spawn with a male with eggs when tested a second time. However, some females were tested twice in the second part of the experiment if they became gravid again (20 females refused males without eggs in the first presentation, yet these females were tested 27 times with a second male; page 156 and Table I). Because the data were not independent, the number of
9 1989 The Association for the Study of Animal Behaviour 859
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females that responded as Ridley & Rechten had predicted was inflated. In addition to these problems with the analysis, female sticklebacks in the wild are more likely to come in contact with territorial males simultaneously during courtship rather than sequentially and in isolation (Wootton 1976). Any effects due to male-male competition during courtship could not be detected in Ridley & Rechten's experiment. Therefore a simultaneous choice experiment was conducted to determine the relative importance of eggs in a male's nest with respect to female mate choice in three-spined sticklebacks. Rohwer (1978) and Ridley & Rechten (1981) proposed that if the probability of egg survival increases as the total number of eggs in a nest increases, natural selection should favour those females who preferentially spawn in nests that already contain eggs. Two predictions that differ on relative strength follow from an experiment where a female has a choice between two males, one with and one without eggs in his nest. If the relative number of eggs in a nest is the criterion of choice as implied by the functional hypothesis, the strongest prediction one could make is that females should visit both nests before spawning in the one with more eggs. A weaker prediction is that females should follow either male back to his nest, but be more likely to spawn with the male with eggs in his nest, if the presence or absence of eggs in a nest is the criterion of choice. In both cases it is assumed that territory size, nest site quality and male quality are controlled.
METHODS Collection and Maintenance of Fish Three-spined sticklebacks were seined from Priest Creek, Quebec, (45 48' N, 75 43' W) on 19 October 1987. The fish were transferred directly to
holding tanks in the Biology Department of Queen's University. The males (55) and females (75) were eventually separated and held in flowthrough aquaria measuring 85 x 45 x 30 cm. The fish were put on an 8:16 h light:dark cycle and maintained at 10~ Four males and eight females were transferred to a separate room on 3 December and put on a L : D 16:8 h cycle and water temperature was gradually increased to 18~ Once the males started building nests and the females became gravid, the light and water temperature were increased as above for the remaining fish in the holding tanks on 4 January. Fish were fed frozen brine shrimp twice a day.
Experimental Procedure A large tank measuring 120 x 45 x 45 cm was used in the mate choice tests. It was housed in an adjoining room to the holding tanks and was maintained on a L : D 16:8 h cycle and t 8~ The tank was separated into two equal areas by an opaque divider and a removable partition (Fig. 1). The removable partition could be operated from behind a blind by a pulley system. Two identical plastic plants and some large rocks were placed in the corner and on either side of the divider. This discouraged the males from nesting in these corners and also served as cover for the fish. Two charcoal filters were set in the opposite back corners. Sand was scattered in front of the filters and a small rock placed at the back of the tank and next to the filter. The males invariably built their nests between the rock and the filter. Equal proportions of nesting material were scattered on the floor in each half of the tank. Two males displaying breeding colours and of approximately the same size were placed in either side of the partition in the experimental tank and allowed to build their nests. Females were kept in the holding tanks and used as they became gravid.
Filter ~) Nest 0
Rock
~1~ Plant -:': Sand
Figure 1. View of the experimental tank from above.
Jamieson & Colgan: Mate choice in sticklebacks Females were tested for receptivity to male courtship by exposing them to a nesting male in a separate tank. Only those females that showed the characteristic 'head-up' and following behaviour (Tinbergen 1951) within 10 s of being contacted by the test male were used in a subsequent trial. To set up a choice trial, one of the two males was chosen to be 'with eggs' by the flip of a coin. A receptive female was put into a clear plastic container measuring 14 x 8 x 7 cm. The container had a removable top attached to monofilament fishing line that was held by the observer from behind a blind. The container with the female was placed on the floor of the tank and inside the territory of the male designated to be 'without eggs'. With the partition in place, the male was allowed to court the female for 3 rain. The container was then removed and placed inside the territory of the male designated to be 'with eggs'. The male was allowed to court for 3 min before the top was removed, The female was then free to swim out and spawn with the male. In this way, both males had courtship experience prior to a choice trial. A choice trial using another gravid female was conducted 4-24 h after one of the males had received eggs (hereafter referred to as an 'egg' trial). This was within the 1-96-h period in which males will court gravid females after spawning (Wootton 1976). Before the female was introduced, the partition was removed to allow the two males to interact and establish their territorial boundary. This procedure resulted in less direct aggression between the two males during the choice trial. The container containing the gravid female was placed in the middle of the tank on the territorial boundary between the two males. The number of 'zigzags' directed at the female and number of attacks directed at the other male were recorded for both males while the female was in the container. Any difference in brightness between the two males was also recorded and scored on a relative basis. The top was removed 3 rain after both males had initially approached the female. The trial ended either when the female spawned and left the nest or when 30 rain had elapsed. After a spawning, all eggs were removed and the nests of both males destroyed. The procedure was repeated once the males had renested except the other male was now 'with eggs'. This design was implemented to control for male quality such as differences in size. After the completion of the second choice trial,
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the two males were removed, their standard lengths recorded to the nearest mm, and two new males introduced. Females were used only once for the choice trials. They could, however, be reused in egg trials when they became gravid again. All trials were videotaped from behind a blind. The frequency of various responses of the males and the female was scored during playback of the tape. The simultaneous choice trials commenced 21 January and concluded 20 April 1988. A sequential choice experiment was also conducted to determine whether females were more likely to abort entry into nests without eggs. The procedures for this experiment were the same as those used in the simultaneous choice test except the female was released with the male without any eggs and whose territory was isolated by a movable partition from another male with eggs. If the female aborted entry into the nest of the male without eggs, the partition was raised and she was free to swim into the other male's territory. At the beginning of a test when neither male had eggs in his nest, the introduction of a female into one of the territories was again designated as an 'egg' trial. If the female spawned with the male, a second female was released with the other male and this qualified as a 'choice' trial. However, if the female aborted nest entry during an egg trial, she was removed and tested later that day with the same male in a choice trial. The males were used in two choice trials, once with and once without eggs, before being replaced by two new males. The sequential choice trials commenced 22 April and ended 26 May.
RESULTS Simultaneous Choice Test In 22 of 26 (85%) choice trials, the female spawned in the nest of the male with eggs (binomial test; P < 0.001). However, mate choice on the basis of a female actively avoiding spawning in a nest without eggs could be attributed to only four of 26 (15%) trials. That is, in only four trials did a female follow the male without eggs back to his nest and exhibit the behaviour of poking her snout one to three times into the nest entrance before swimming away and subsequently spawning with the male who had eggs. In four other trials, the female spawned with a male without eggs in his nest. Furthermore, there was no indication of females visiting nests before spawning took place. It
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Animal Behaviour, 38, 5
appeared that in most cases males with eggs were simply more successful at leading females to their nests. Therefore we asked the question: what is it about males with eggs in their nest that allows them to achieve a higher number of spawn• There were no significant effects due to the order in which males in paired trials spawned for any of the variables examined below. Because males were tested twice, once with and once without eggs, physical attributes such as differences in size could not account for the skew in mating success in the direction of males with eggs. The larger male spawned in 13 trials, the smaller in nine O?• difference in size between m a l e s = 2 . 3 +0.50 mm, N = 13; Witcoxon matched-pairs, z = - 0 . 5 8 , N = 2 2 , P>0-10). There was no difference in size between the two males in four trials. Other attributes of males such as brightness of colour, aggression and courtship intensity were more variable between trials. We examined whether there was a relationship between a male having eggs in his nest and the expression of these traits, relative to the male without eggs, during the first 3" min of a trial when the female was still confined to the clear container. There was no significant difference in the number of attacks initiated by males with eggs ()?•177 or without eggs (2.7• N = 2 6 ; Wilcoxon matched-pairs, z = - 0 . 5 2 , N = 23, P > 0-10). However, males with eggs in their nest were more often brighter than males without eggs (14 and four trials, respectively, sign test, P=0"03; there were eight trials in which no difference in brightness could be detected). Males with eggs also performed more 'zig-zags' while approaching the female in the container ()?•177 than males without eggs in their nest (4.9• N = 2 6 ) , although the difference was not quite significant (Wilcoxon matchedpairs, z = - 1 ' 5 3 , N = 2 3 , P=0"06). Despite the initial tendency for males with eggs to be brighter and to exhibit more zig-zags, females showed no preference for males with or without eggs in their nests as measured by the territories females first entered when released (13 and 13 trials, respectively, binomial test, P > 0 . 1 0 ) , or by the males they first courted (14 and 12, respectively, P > 0-10). In fact, females were very passive in their courtship behaviour; they usually approached and courted with whichever male happened to nip or bite them. Overall, females did not court more frequently with males with eggs
Table I. The number of first follows by females of males with and without eggs in their nest No. of trials Males Males with eggs without eggs Female follows directly to nest after release Female courts and then follows to nest Female courts both males and then follows to nest
10
l
Totals
21
5
3
0
8
4
()?+SE=2"2+0"62) than males without eggs in their nest (3'2• N = 2 6 ; Wilcoxon matchedpairs, z = - 0 . 0 2 , N = 2 1 , P > 0 . 1 0 ) , nor did they spend more time courting males with eggs (28.5_ 9.28 s) than males without eggs (38.5 _+ 12.9 s, N = 2 6 ; Wilcoxon matched-pairs, z = - 0 ' 5 0 , N = 2 5 , P > 0.10). Although not all 'follows' to the nest by females result in spawnings, they do give some indication of a males' ability to lead a female to his nest. When we examined which male the female followed first in each of the trials, we found that males with eggs in their nest were followed significantly more often, even after the female had courted with both males (binomial test, N = 2 6 , P < 0 ' 0 0 1 ; Table I). This indicates that males without eggs in their nests were able to attract females into their territories and court with them, but were much less likely to lead females to their nests. This is further illustrated by looking at the events that lead up to a female spawning in a particular nest. In seven trials the female initially went into the territory of the male with eggs in his nest and subsequently spawned; in three trials the same occurred for the male without eggs. In 15 other trials, the female had been courted by the male without eggs in his nest before she left and subsequently spawned with the male with eggs. These departures resulted from females (1) moving towards the male with eggs who was zig-zagging intensely at the border of his territory while the male without eggs was at his nest (four trials), (2) moving away from the male without eggs after he had bitten her during courtship (seven trials), or (3) being chased by the male without eggs after the
Jamieson & Colgan: Mate choice in sticklebacks female had followed him to his nest and aborted nest entry (four trials). With respect to aborted nest entries, there were also two trials in which females aborted entry into nests with eggs, but ended up spawning in these nests later in the same trial. In two other trials, females aborted entry into the nests of both males several times and failed to spawn with either; both females spawned in nests with eggs when retested. In summary, males with eggs in their nest tended to be more brightly coloured, courted more intensely when a female was first introduced and were more successful at leading her to their nest once she was released. They were also more likely to attract a female away from the male without eggs in his nest and less likely to let her leave once she was there. In a few cases, the absence of eggs in a nest directly accounted for a male's failure to achieve a spawning.
I00
o
75
~
5O
~
25
4 402
50
863
8 813121071591055
Z
~
2
4
6
8 IO 12 Week no.
14
16
I 18
Figure 2. The percentage of trials in which aborted entries into nests occurred for each week of the simultaneous and sequential choice experiments. The number of trials run each week is shown at the top of the graph.
could be retested. When tested again in a choice trial the following week, she aborted once, then spawned with the male without eggs. Seasonal Pattern of Mate Choice
Sequential Choice Test Because relatively few females in the simultaneous choice test were actually led to nests that had no eggs, many more females might have aborted nest entries if given the opportunity. In the egg trials (when a female was introduced to a male in isolation), 14 of 38 (37%) females aborted nest entries as many as three times before being replaced by another female. Seven of these females had previously been tested in a choice trial and all had spawned in nests with eggs without having inspected a nest with no eggs. Therefore, with the use of a movable partition, we conducted a sequential choice experiment in which the female was allowed to court a mate with eggs only after she had had the opportunity to mate with a male without eggs in his nest. O f the 32 females tested in the sequential choice trials, 23 (72%) females entered the nest of the male without eggs and spawned (binomial test, P < 0-001). Six females aborted entry into the nest without eggs and subsequently spawned in the nest of the male with eggs. Three of these six aborted entry during a choice trial. The other three aborted during an egg trial and later aborted again and then spawned in a nest with eggs when retested in a choice trial. Two other females aborted during an egg trial, but spawned with the male without eggs when retested in a choice trial. A third female aborted nests of both males during a choice trial and then released her eggs that night before she
There was no indication that females became more or less likely to abort a nest entry as the breeding period progressed. The simultaneous and sequential choice tests ran for a total of 17 weeks and the proportion of trials in which females aborted entries into nests without eggs during both egg and choice trials was relatively constant (Fig. 2).
DISCUSSION Our results indicate that male sticklebacks that have recently spawned and have eggs in their nests are significantly more likely to obtain a second spawning than males that have not spawned and have no eggs in their nests. However, there was little evidence of females visiting nests before spawning and rejecting those males without eggs as would be predicted if females had been selected to spawn preferentially in nests with relatively more eggs. We can not exclude the possibility that females were using olfactory cues from the eggs to base their decisions to spawn with particular males. However, spawning success was more a function of differences in male behaviour than active choice by females. Females responded and courted with whichever male approached and bit them. Over time, males with eggs in their nest were better able to maintain a courting female inside their territory and eventually lead her to their nests. These results
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Animal Behaviour, 38, 5
are similar to those of Ward & FitzGerald (1987) who found that some male sticklebacks obtained more matings as a consequence of variation in male behaviour (with respect to aggressiveness) rather than active female choice. Eggs in a nest could positively influence the female to enter that nest once she is led to it. However, far more females entered and spawned in nests without eggs in the egg trials and the sequential choice trials than aborted. Even in the simultaneous choice trials, an equal number of females spawned or aborted entries in nests without eggs (four trials each). Furthermore, aborted entries were not seen exclusively in nests without eggs. These findings raise further doubt about the conclusion reached by Ridley & Rechten (1981) that female sticklebacks 'prefer' to mate with males with eggs. In addition to the questionable validity of their analysis (see Introduction), by testing only a single male at a time, with or without eggs, they eliminated any important effect that can occur as a result of variation among males in courtship behaviour. For example, in clear contrast to our results, they found no significant difference between males with and without eggs in the proportion of follows by females. It appears that the initial act of spawning or the eggs themselves increases a male's motivation to spawn again. However, why do males without eggs not simply increase their courtship intensity? Certainly one might argue that there should be selection pressure on males without eggs to court more intensely to gain eggs. This argument, however, does not take into account the time scale and constraints over which breeding is occurring. After a male receives his first clutch of eggs, he will enter the parental phase of the reproductive cycle in about 4 days, at which time courtship activity ceases (Wootton 1976). Therefore selection should act on all males to increase courtship activity and obtain as many clutches as they are capable of caring for before the males become parental. Males without eggs are not under such short-term time constraints. Furthermore, the multiple clutches laid by females over a relatively long breeding period ensures that there are gravid females available beyond the first breeding bout. Preliminary results from a study we are presently conducting support the hypothesis that mating success among a small colony of males shows a serial or sequential pattern of egg acquisition. Nevertheless, mating success is not a random
process dependent on whichever male gets the first spawning. Several factors such as territorial size (van den Assem 1967), quality of nest site (Sargent & Gebler 1980; Sargent 1982) and levels of aggression (Ward & FitzGerald 1987), all of which presumably reflect differences in male competitiveness, are known to affect mating success of males before they obtain eggs. However, our study is the first to indicate that any initial advantage a particular male has over other males in attracting a female to his territory and nest will be enhanced once he spawns. For their part, females seem simply to 'follow the path of least resistance' (Sargent 1982; Ward & FitzGerald 1987). It is still interesting that some females showed a distinctive behavioural pattern of poking their heads into a nest and backing out, as if refusing to enter. Wootton (1976) offered the motivational explanation that females were not quite ready to spawn. Ridley & Rechten (1981) countered this by offering the functional explanation that females inspect a nest and are more likely to refuse entry if it is disadvantageous to spawn in nests that lack eggs. They suggested several adaptive advantages to females spawning in nests that already contain eggs. However, in presenting this argument Ridley & Rechten (1981) did not emphasize the fact that in their study 49% of the females that followed males with eggs also aborted nest entry, and aborted a second time when retested (see their Table 1). Similarly, we found that the absence of eggs in a male's nest was not a good predictor of when a female would abort entry. These observations are more consistent with Wootton's (1976) explanation. When females are tested with receptive males at various times in the interval between successive spawnings, courting and following behaviour are exhibited much earlier than behaviour associated with entering the nest (Wootton 1974). In fact, entrance into a male's nest only appears shortly before spawning actually takes place. Therefore, many of the aborted nest entries observed in the laboratory may be an artefact of using females that are not quite ready to spawn. We are now working with an experimental design that allows gravid females to enter and leave a colony of territorial males on their own accord. In this way we hope to reduce potential procedural artefacts in a controlled experiment so that the factors that affect mating success can be determined. In the meantime, we suggest that the
Jamieson & Colgan: M a t e choice in sticklebacks conclusion t h a t female sticklebacks prefer to m a t e with males with eggs in their nests should be viewed with caution.
ACKNOWLEDGMENTS W e t h a n k B. C o a d for assistance in collecting the fish a n d L. J a m i e s o n a n d J. F r a m e for technical assistance. V. (3otceitas p r o v i d e d helpful suggestions d u r i n g the setting u p o f the experiments. T h e feedback o n the day-to-day results from P. K r a n nitz d u r i n g the evening walks h o m e was very m u c h appreciated. We would also like to t h a n k (3. F i t z G e r a l d and his g r a d u a t e students a n d the gang at the B. E. E. R. S. Weekly Seminars for their discussion of the results, P. J o h a n s e n , I. Jones, M. Ridgway a n d C. Sargent p r o v i d e d helpful comm e n t s o n an earlier draft o f the manuscript. O u r research was funded by a N S E R C P o s t d o c t o r a l Fellowship (I.(3.J.) a n d N S E R C O p e r a t i n g G r a n t (P.W.C.).
REFERENCES van den Assem, J. 1976. Territoriality in the three-spined stickleback, Gasterosteus aculeatus L., an experimental study in intra-specific aggression. Behav., Suppl., 16, 1164. Bateson, P. P. G. (Ed.) 1983. Mate Choice. New York: Cambridge University Press. Constanz, G. D. 1985. Allopaternal care in the tessellated darter, Etheostoma olmstedi (Pisces: Percidae), Environ. Biol. Fish., 14, 175-183. FitzGerald, G. J. & Wootton, R. J. 1986. The behavioural ecology of sticklebacks In: The Behaviour of Teleost Fishes (Ed. by T. J. Pitcher), pp. 409-432. London: Croom Helm.
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Halliday, T. R. 1983. The study of mate choice. In: Mate Choice (Ed. by P. P. G. Bateson), pp. 3-32. New York: Cambridge University Press. Marconato, A. & Bisazza, A. 1986. Males whose nests contain eggs are preferred by female Cottus gobio L. (Pisces, Cottidae). Anita. Behav., 34, 1580-1582. Ridley, M. & Rechten, C. 1981. Female sticklebacks prefer to spawn with males whose nests contain eggs. Behaviour, 76, 152-161. Rohwer, S. 1978. Parent cannibalism of offspring and egg raiding as a courtship strategy. Am. Nat., 112, 429 440. Sargent, R. C. 1982. Territory quality, male quality, courtship intrusion, and female nest-choice in the threespine stickleback, Gasterosteus aculeatus. Anim. Behav., 30, 364-374. Sargent, R. C. & Gebler, J. B. 1980. Effects of nest site concealment on hatching success, reproductive success, and paternal behavior of the threespine stickleback, Gasterosteus aeuleatus. Behav. Ecol. Sociobiol., 7, 137 142. Tinbergen, N. 1951. The Study of Instinct. Oxford: Clarendon Press. Unger, L. M. & Sargent, R. C. 1988. Allopaternal care in the fathead minnow, Pimephales promelas: females prefer males with eggs. Behav. Ecol. Sociobiol., 23, 2732. Vickery, W. L., Whoriskey, F. G. & FitzGerald, G. J. 1988. On the evolution of nest raiding and male defensive behaviour in sticklebacks (Pisces: Gasterosteidae). Behav. Ecol. Sociobiol., 22, 185-193. Ward, G. & FitzGerald, G. J. 1987. Male aggression and female choice in the threespine stickleback, Gasterosteus aeuleatus L. J. Fish. Biol., 30, 679-690. Wootton, R. J. 1974. Changes in the courtship behaviour of female three-spined sticklebacks between spawnings. Anim. Behav., 22, 850-855. Wootton, R. J. 1976. The Biology of Sticklebacks. New York: Academic Press. (Received 7 October 1988; revised 26 January 1989; MS. number: A 5402)
Eggs in the nests of males and their effect on mate choice in the three-spined stickleback I A N G. J A M 1 E S O N & P A T R I C K W. C O L G A N
Department of Biology, Queen's University, Kingston, Ontario, K7L 3N6, Canada
Abstract. It is thought that female three-spined sticklebacks, Gasterosteus aculeatus, prefer to mate with males whose nests already contain eggs. This is based on the results from a study where gravid females were tested sequentially, first with a male without eggs, and then with a male with eggs. However, that experimental design fails to detect important effects due to male-male competition. Simultaneous and sequential choice experiments were conducted to determine the relative importance of eggs in a nest with respect to female mate choice. In the simultaneous choice tests, males with eggs in their nest were more likely to obtain a second spawning than males without eggs. However, there was little evidence of females actively 'choosing' mates on the basis of presence or absence of eggs. Rather, males that had recently spawned courted more intensely and were more successful at leading females to their nests. In only a few cases could a male's failure to spawn be attributed to a female aborting entry into a nest without eggs. In the sequential choice tests, significantly more females entered and spawned than aborted entry into nests without eggs. Furthermore, aborted entries were not seen exclusively in nests without eggs. The results indicated that the presence of eggs had a general priming effect on courtship behaviour of males, but evidence for active female choice was lacking. Overall, females did not exhibit the behavioural patterns predicted from the hypothesis that females should spawn preferentially in nests with eggs.
The importance of male traits with respect to female mate choice has recently received a great deal of attention (e.g. Bateson 1983). Female preferences for particular traits can result in variation in male mating success. However, it has been notoriously difficult to distinguish the effects of active female choice from those of passive choice in intrasexual competition (Halliday 1983). In fishes with exclusively paternal care, several possible adaptive advantages have been suggested for females choosing males who are already mated (see Rohwer 1978; Ridley & Rechten 1981). There is now evidence in four species that females prefer to spawn in nests that already contain eggs. In three of these species, the tessellated darter, Etheostoma olmstedi (Constanz 1985), the river bullhead, Cottus gobio (Marconato & Bisazza 1986), and the fathead minnow, Pimephales promelas (Unger & Sargent 1988), males defend rocks and crevices, but show little or no courtship behaviour toward gravid females. Females inspect suitable spawning sites defended by the males and nests that already contain eggs receive significantly more new eggs than nests with no eggs. By contrast, male three-spined sticklebacks, Gasterosteus aculeatus, construct a substrate nest out of. vegetation and perform elaborate courtship 0003-3472/89/110859+ 07 $03.00/0
behaviour in order to lead gravid females to their nests. Ridley & Rechten (1981) conducted a sequential choice experiment in which females were presented to single males in isolation. Some females were more likely to enter a nest and spawn than refuse mating if the nest of the male already contained eggs; a refusal took the form of a female putting her snout into the nest and then backing out and swimming away. This study has often been cited as evidence that female sticklebacks prefer to spawn in nests with eggs (e.g. Sargent 1982; Halliday 1983; Fitzgerald & Wootton 1986; Vickcry et al. 1988), yet such a conclusion is difficult to draw from Ridley & Rechten's results. Their initial experiment found that the presence of eggs in a nest did not significantly increase the female's probability of spawning (Ridley & Rechten 1981). In a second experiment they reported that some of the females who had refused to spawn in a nest of a male without eggs were more likely to spawn with a male with eggs when tested a second time. However, some females were tested twice in the second part of the experiment if they became gravid again (20 females refused males without eggs in the first presentation, yet these females were tested 27 times with a second male; page 156 and Table I). Because the data were not independent, the number of
9 1989 The Association for the Study of Animal Behaviour 859
Animal Behaviour, 38, 5
860
females that responded as Ridley & Rechten had predicted was inflated. In addition to these problems with the analysis, female sticklebacks in the wild are more likely to come in contact with territorial males simultaneously during courtship rather than sequentially and in isolation (Wootton 1976). Any effects due to male-male competition during courtship could not be detected in Ridley & Rechten's experiment. Therefore a simultaneous choice experiment was conducted to determine the relative importance of eggs in a male's nest with respect to female mate choice in three-spined sticklebacks. Rohwer (1978) and Ridley & Rechten (1981) proposed that if the probability of egg survival increases as the total number of eggs in a nest increases, natural selection should favour those females who preferentially spawn in nests that already contain eggs. Two predictions that differ on relative strength follow from an experiment where a female has a choice between two males, one with and one without eggs in his nest. If the relative number of eggs in a nest is the criterion of choice as implied by the functional hypothesis, the strongest prediction one could make is that females should visit both nests before spawning in the one with more eggs. A weaker prediction is that females should follow either male back to his nest, but be more likely to spawn with the male with eggs in his nest, if the presence or absence of eggs in a nest is the criterion of choice. In both cases it is assumed that territory size, nest site quality and male quality are controlled.
METHODS Collection and Maintenance of Fish Three-spined sticklebacks were seined from Priest Creek, Quebec, (45 48' N, 75 43' W) on 19 October 1987. The fish were transferred directly to
holding tanks in the Biology Department of Queen's University. The males (55) and females (75) were eventually separated and held in flowthrough aquaria measuring 85 x 45 x 30 cm. The fish were put on an 8:16 h light:dark cycle and maintained at 10~ Four males and eight females were transferred to a separate room on 3 December and put on a L : D 16:8 h cycle and water temperature was gradually increased to 18~ Once the males started building nests and the females became gravid, the light and water temperature were increased as above for the remaining fish in the holding tanks on 4 January. Fish were fed frozen brine shrimp twice a day.
Experimental Procedure A large tank measuring 120 x 45 x 45 cm was used in the mate choice tests. It was housed in an adjoining room to the holding tanks and was maintained on a L : D 16:8 h cycle and t 8~ The tank was separated into two equal areas by an opaque divider and a removable partition (Fig. 1). The removable partition could be operated from behind a blind by a pulley system. Two identical plastic plants and some large rocks were placed in the corner and on either side of the divider. This discouraged the males from nesting in these corners and also served as cover for the fish. Two charcoal filters were set in the opposite back corners. Sand was scattered in front of the filters and a small rock placed at the back of the tank and next to the filter. The males invariably built their nests between the rock and the filter. Equal proportions of nesting material were scattered on the floor in each half of the tank. Two males displaying breeding colours and of approximately the same size were placed in either side of the partition in the experimental tank and allowed to build their nests. Females were kept in the holding tanks and used as they became gravid.
Filter ~) Nest 0
Rock
~1~ Plant -:': Sand
Figure 1. View of the experimental tank from above.
Jamieson & Colgan: Mate choice in sticklebacks Females were tested for receptivity to male courtship by exposing them to a nesting male in a separate tank. Only those females that showed the characteristic 'head-up' and following behaviour (Tinbergen 1951) within 10 s of being contacted by the test male were used in a subsequent trial. To set up a choice trial, one of the two males was chosen to be 'with eggs' by the flip of a coin. A receptive female was put into a clear plastic container measuring 14 x 8 x 7 cm. The container had a removable top attached to monofilament fishing line that was held by the observer from behind a blind. The container with the female was placed on the floor of the tank and inside the territory of the male designated to be 'without eggs'. With the partition in place, the male was allowed to court the female for 3 rain. The container was then removed and placed inside the territory of the male designated to be 'with eggs'. The male was allowed to court for 3 min before the top was removed, The female was then free to swim out and spawn with the male. In this way, both males had courtship experience prior to a choice trial. A choice trial using another gravid female was conducted 4-24 h after one of the males had received eggs (hereafter referred to as an 'egg' trial). This was within the 1-96-h period in which males will court gravid females after spawning (Wootton 1976). Before the female was introduced, the partition was removed to allow the two males to interact and establish their territorial boundary. This procedure resulted in less direct aggression between the two males during the choice trial. The container containing the gravid female was placed in the middle of the tank on the territorial boundary between the two males. The number of 'zigzags' directed at the female and number of attacks directed at the other male were recorded for both males while the female was in the container. Any difference in brightness between the two males was also recorded and scored on a relative basis. The top was removed 3 rain after both males had initially approached the female. The trial ended either when the female spawned and left the nest or when 30 rain had elapsed. After a spawning, all eggs were removed and the nests of both males destroyed. The procedure was repeated once the males had renested except the other male was now 'with eggs'. This design was implemented to control for male quality such as differences in size. After the completion of the second choice trial,
861
the two males were removed, their standard lengths recorded to the nearest mm, and two new males introduced. Females were used only once for the choice trials. They could, however, be reused in egg trials when they became gravid again. All trials were videotaped from behind a blind. The frequency of various responses of the males and the female was scored during playback of the tape. The simultaneous choice trials commenced 21 January and concluded 20 April 1988. A sequential choice experiment was also conducted to determine whether females were more likely to abort entry into nests without eggs. The procedures for this experiment were the same as those used in the simultaneous choice test except the female was released with the male without any eggs and whose territory was isolated by a movable partition from another male with eggs. If the female aborted entry into the nest of the male without eggs, the partition was raised and she was free to swim into the other male's territory. At the beginning of a test when neither male had eggs in his nest, the introduction of a female into one of the territories was again designated as an 'egg' trial. If the female spawned with the male, a second female was released with the other male and this qualified as a 'choice' trial. However, if the female aborted nest entry during an egg trial, she was removed and tested later that day with the same male in a choice trial. The males were used in two choice trials, once with and once without eggs, before being replaced by two new males. The sequential choice trials commenced 22 April and ended 26 May.
RESULTS Simultaneous Choice Test In 22 of 26 (85%) choice trials, the female spawned in the nest of the male with eggs (binomial test; P < 0.001). However, mate choice on the basis of a female actively avoiding spawning in a nest without eggs could be attributed to only four of 26 (15%) trials. That is, in only four trials did a female follow the male without eggs back to his nest and exhibit the behaviour of poking her snout one to three times into the nest entrance before swimming away and subsequently spawning with the male who had eggs. In four other trials, the female spawned with a male without eggs in his nest. Furthermore, there was no indication of females visiting nests before spawning took place. It
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Animal Behaviour, 38, 5
appeared that in most cases males with eggs were simply more successful at leading females to their nests. Therefore we asked the question: what is it about males with eggs in their nest that allows them to achieve a higher number of spawn• There were no significant effects due to the order in which males in paired trials spawned for any of the variables examined below. Because males were tested twice, once with and once without eggs, physical attributes such as differences in size could not account for the skew in mating success in the direction of males with eggs. The larger male spawned in 13 trials, the smaller in nine O?• difference in size between m a l e s = 2 . 3 +0.50 mm, N = 13; Witcoxon matched-pairs, z = - 0 . 5 8 , N = 2 2 , P>0-10). There was no difference in size between the two males in four trials. Other attributes of males such as brightness of colour, aggression and courtship intensity were more variable between trials. We examined whether there was a relationship between a male having eggs in his nest and the expression of these traits, relative to the male without eggs, during the first 3" min of a trial when the female was still confined to the clear container. There was no significant difference in the number of attacks initiated by males with eggs ()?•177 or without eggs (2.7• N = 2 6 ; Wilcoxon matched-pairs, z = - 0 . 5 2 , N = 23, P > 0-10). However, males with eggs in their nest were more often brighter than males without eggs (14 and four trials, respectively, sign test, P=0"03; there were eight trials in which no difference in brightness could be detected). Males with eggs also performed more 'zig-zags' while approaching the female in the container ()?•177 than males without eggs in their nest (4.9• N = 2 6 ) , although the difference was not quite significant (Wilcoxon matchedpairs, z = - 1 ' 5 3 , N = 2 3 , P=0"06). Despite the initial tendency for males with eggs to be brighter and to exhibit more zig-zags, females showed no preference for males with or without eggs in their nests as measured by the territories females first entered when released (13 and 13 trials, respectively, binomial test, P > 0 . 1 0 ) , or by the males they first courted (14 and 12, respectively, P > 0-10). In fact, females were very passive in their courtship behaviour; they usually approached and courted with whichever male happened to nip or bite them. Overall, females did not court more frequently with males with eggs
Table I. The number of first follows by females of males with and without eggs in their nest No. of trials Males Males with eggs without eggs Female follows directly to nest after release Female courts and then follows to nest Female courts both males and then follows to nest
10
l
Totals
21
5
3
0
8
4
()?+SE=2"2+0"62) than males without eggs in their nest (3'2• N = 2 6 ; Wilcoxon matchedpairs, z = - 0 . 0 2 , N = 2 1 , P > 0 . 1 0 ) , nor did they spend more time courting males with eggs (28.5_ 9.28 s) than males without eggs (38.5 _+ 12.9 s, N = 2 6 ; Wilcoxon matched-pairs, z = - 0 ' 5 0 , N = 2 5 , P > 0.10). Although not all 'follows' to the nest by females result in spawnings, they do give some indication of a males' ability to lead a female to his nest. When we examined which male the female followed first in each of the trials, we found that males with eggs in their nest were followed significantly more often, even after the female had courted with both males (binomial test, N = 2 6 , P < 0 ' 0 0 1 ; Table I). This indicates that males without eggs in their nests were able to attract females into their territories and court with them, but were much less likely to lead females to their nests. This is further illustrated by looking at the events that lead up to a female spawning in a particular nest. In seven trials the female initially went into the territory of the male with eggs in his nest and subsequently spawned; in three trials the same occurred for the male without eggs. In 15 other trials, the female had been courted by the male without eggs in his nest before she left and subsequently spawned with the male with eggs. These departures resulted from females (1) moving towards the male with eggs who was zig-zagging intensely at the border of his territory while the male without eggs was at his nest (four trials), (2) moving away from the male without eggs after he had bitten her during courtship (seven trials), or (3) being chased by the male without eggs after the
Jamieson & Colgan: Mate choice in sticklebacks female had followed him to his nest and aborted nest entry (four trials). With respect to aborted nest entries, there were also two trials in which females aborted entry into nests with eggs, but ended up spawning in these nests later in the same trial. In two other trials, females aborted entry into the nests of both males several times and failed to spawn with either; both females spawned in nests with eggs when retested. In summary, males with eggs in their nest tended to be more brightly coloured, courted more intensely when a female was first introduced and were more successful at leading her to their nest once she was released. They were also more likely to attract a female away from the male without eggs in his nest and less likely to let her leave once she was there. In a few cases, the absence of eggs in a nest directly accounted for a male's failure to achieve a spawning.
I00
o
75
~
5O
~
25
4 402
50
863
8 813121071591055
Z
~
2
4
6
8 IO 12 Week no.
14
16
I 18
Figure 2. The percentage of trials in which aborted entries into nests occurred for each week of the simultaneous and sequential choice experiments. The number of trials run each week is shown at the top of the graph.
could be retested. When tested again in a choice trial the following week, she aborted once, then spawned with the male without eggs. Seasonal Pattern of Mate Choice
Sequential Choice Test Because relatively few females in the simultaneous choice test were actually led to nests that had no eggs, many more females might have aborted nest entries if given the opportunity. In the egg trials (when a female was introduced to a male in isolation), 14 of 38 (37%) females aborted nest entries as many as three times before being replaced by another female. Seven of these females had previously been tested in a choice trial and all had spawned in nests with eggs without having inspected a nest with no eggs. Therefore, with the use of a movable partition, we conducted a sequential choice experiment in which the female was allowed to court a mate with eggs only after she had had the opportunity to mate with a male without eggs in his nest. O f the 32 females tested in the sequential choice trials, 23 (72%) females entered the nest of the male without eggs and spawned (binomial test, P < 0-001). Six females aborted entry into the nest without eggs and subsequently spawned in the nest of the male with eggs. Three of these six aborted entry during a choice trial. The other three aborted during an egg trial and later aborted again and then spawned in a nest with eggs when retested in a choice trial. Two other females aborted during an egg trial, but spawned with the male without eggs when retested in a choice trial. A third female aborted nests of both males during a choice trial and then released her eggs that night before she
There was no indication that females became more or less likely to abort a nest entry as the breeding period progressed. The simultaneous and sequential choice tests ran for a total of 17 weeks and the proportion of trials in which females aborted entries into nests without eggs during both egg and choice trials was relatively constant (Fig. 2).
DISCUSSION Our results indicate that male sticklebacks that have recently spawned and have eggs in their nests are significantly more likely to obtain a second spawning than males that have not spawned and have no eggs in their nests. However, there was little evidence of females visiting nests before spawning and rejecting those males without eggs as would be predicted if females had been selected to spawn preferentially in nests with relatively more eggs. We can not exclude the possibility that females were using olfactory cues from the eggs to base their decisions to spawn with particular males. However, spawning success was more a function of differences in male behaviour than active choice by females. Females responded and courted with whichever male approached and bit them. Over time, males with eggs in their nest were better able to maintain a courting female inside their territory and eventually lead her to their nests. These results
864
Animal Behaviour, 38, 5
are similar to those of Ward & FitzGerald (1987) who found that some male sticklebacks obtained more matings as a consequence of variation in male behaviour (with respect to aggressiveness) rather than active female choice. Eggs in a nest could positively influence the female to enter that nest once she is led to it. However, far more females entered and spawned in nests without eggs in the egg trials and the sequential choice trials than aborted. Even in the simultaneous choice trials, an equal number of females spawned or aborted entries in nests without eggs (four trials each). Furthermore, aborted entries were not seen exclusively in nests without eggs. These findings raise further doubt about the conclusion reached by Ridley & Rechten (1981) that female sticklebacks 'prefer' to mate with males with eggs. In addition to the questionable validity of their analysis (see Introduction), by testing only a single male at a time, with or without eggs, they eliminated any important effect that can occur as a result of variation among males in courtship behaviour. For example, in clear contrast to our results, they found no significant difference between males with and without eggs in the proportion of follows by females. It appears that the initial act of spawning or the eggs themselves increases a male's motivation to spawn again. However, why do males without eggs not simply increase their courtship intensity? Certainly one might argue that there should be selection pressure on males without eggs to court more intensely to gain eggs. This argument, however, does not take into account the time scale and constraints over which breeding is occurring. After a male receives his first clutch of eggs, he will enter the parental phase of the reproductive cycle in about 4 days, at which time courtship activity ceases (Wootton 1976). Therefore selection should act on all males to increase courtship activity and obtain as many clutches as they are capable of caring for before the males become parental. Males without eggs are not under such short-term time constraints. Furthermore, the multiple clutches laid by females over a relatively long breeding period ensures that there are gravid females available beyond the first breeding bout. Preliminary results from a study we are presently conducting support the hypothesis that mating success among a small colony of males shows a serial or sequential pattern of egg acquisition. Nevertheless, mating success is not a random
process dependent on whichever male gets the first spawning. Several factors such as territorial size (van den Assem 1967), quality of nest site (Sargent & Gebler 1980; Sargent 1982) and levels of aggression (Ward & FitzGerald 1987), all of which presumably reflect differences in male competitiveness, are known to affect mating success of males before they obtain eggs. However, our study is the first to indicate that any initial advantage a particular male has over other males in attracting a female to his territory and nest will be enhanced once he spawns. For their part, females seem simply to 'follow the path of least resistance' (Sargent 1982; Ward & FitzGerald 1987). It is still interesting that some females showed a distinctive behavioural pattern of poking their heads into a nest and backing out, as if refusing to enter. Wootton (1976) offered the motivational explanation that females were not quite ready to spawn. Ridley & Rechten (1981) countered this by offering the functional explanation that females inspect a nest and are more likely to refuse entry if it is disadvantageous to spawn in nests that lack eggs. They suggested several adaptive advantages to females spawning in nests that already contain eggs. However, in presenting this argument Ridley & Rechten (1981) did not emphasize the fact that in their study 49% of the females that followed males with eggs also aborted nest entry, and aborted a second time when retested (see their Table 1). Similarly, we found that the absence of eggs in a male's nest was not a good predictor of when a female would abort entry. These observations are more consistent with Wootton's (1976) explanation. When females are tested with receptive males at various times in the interval between successive spawnings, courting and following behaviour are exhibited much earlier than behaviour associated with entering the nest (Wootton 1974). In fact, entrance into a male's nest only appears shortly before spawning actually takes place. Therefore, many of the aborted nest entries observed in the laboratory may be an artefact of using females that are not quite ready to spawn. We are now working with an experimental design that allows gravid females to enter and leave a colony of territorial males on their own accord. In this way we hope to reduce potential procedural artefacts in a controlled experiment so that the factors that affect mating success can be determined. In the meantime, we suggest that the
Jamieson & Colgan: M a t e choice in sticklebacks conclusion t h a t female sticklebacks prefer to m a t e with males with eggs in their nests should be viewed with caution.
ACKNOWLEDGMENTS W e t h a n k B. C o a d for assistance in collecting the fish a n d L. J a m i e s o n a n d J. F r a m e for technical assistance. V. (3otceitas p r o v i d e d helpful suggestions d u r i n g the setting u p o f the experiments. T h e feedback o n the day-to-day results from P. K r a n nitz d u r i n g the evening walks h o m e was very m u c h appreciated. We would also like to t h a n k (3. F i t z G e r a l d and his g r a d u a t e students a n d the gang at the B. E. E. R. S. Weekly Seminars for their discussion of the results, P. J o h a n s e n , I. Jones, M. Ridgway a n d C. Sargent p r o v i d e d helpful comm e n t s o n an earlier draft o f the manuscript. O u r research was funded by a N S E R C P o s t d o c t o r a l Fellowship (I.(3.J.) a n d N S E R C O p e r a t i n g G r a n t (P.W.C.).
REFERENCES van den Assem, J. 1976. Territoriality in the three-spined stickleback, Gasterosteus aculeatus L., an experimental study in intra-specific aggression. Behav., Suppl., 16, 1164. Bateson, P. P. G. (Ed.) 1983. Mate Choice. New York: Cambridge University Press. Constanz, G. D. 1985. Allopaternal care in the tessellated darter, Etheostoma olmstedi (Pisces: Percidae), Environ. Biol. Fish., 14, 175-183. FitzGerald, G. J. & Wootton, R. J. 1986. The behavioural ecology of sticklebacks In: The Behaviour of Teleost Fishes (Ed. by T. J. Pitcher), pp. 409-432. London: Croom Helm.
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Halliday, T. R. 1983. The study of mate choice. In: Mate Choice (Ed. by P. P. G. Bateson), pp. 3-32. New York: Cambridge University Press. Marconato, A. & Bisazza, A. 1986. Males whose nests contain eggs are preferred by female Cottus gobio L. (Pisces, Cottidae). Anita. Behav., 34, 1580-1582. Ridley, M. & Rechten, C. 1981. Female sticklebacks prefer to spawn with males whose nests contain eggs. Behaviour, 76, 152-161. Rohwer, S. 1978. Parent cannibalism of offspring and egg raiding as a courtship strategy. Am. Nat., 112, 429 440. Sargent, R. C. 1982. Territory quality, male quality, courtship intrusion, and female nest-choice in the threespine stickleback, Gasterosteus aculeatus. Anim. Behav., 30, 364-374. Sargent, R. C. & Gebler, J. B. 1980. Effects of nest site concealment on hatching success, reproductive success, and paternal behavior of the threespine stickleback, Gasterosteus aeuleatus. Behav. Ecol. Sociobiol., 7, 137 142. Tinbergen, N. 1951. The Study of Instinct. Oxford: Clarendon Press. Unger, L. M. & Sargent, R. C. 1988. Allopaternal care in the fathead minnow, Pimephales promelas: females prefer males with eggs. Behav. Ecol. Sociobiol., 23, 2732. Vickery, W. L., Whoriskey, F. G. & FitzGerald, G. J. 1988. On the evolution of nest raiding and male defensive behaviour in sticklebacks (Pisces: Gasterosteidae). Behav. Ecol. Sociobiol., 22, 185-193. Ward, G. & FitzGerald, G. J. 1987. Male aggression and female choice in the threespine stickleback, Gasterosteus aeuleatus L. J. Fish. Biol., 30, 679-690. Wootton, R. J. 1974. Changes in the courtship behaviour of female three-spined sticklebacks between spawnings. Anim. Behav., 22, 850-855. Wootton, R. J. 1976. The Biology of Sticklebacks. New York: Academic Press. (Received 7 October 1988; revised 26 January 1989; MS. number: A 5402)