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Learned mate recognition and reproductive isolation in guppies
ANIMAL BEHAVIOUR, 2004, 67, 1077e1082 doi:10.1016/j.anbehav.2003.10.010
Learned mate recognition and reproductive isolation in guppies AN NE E. MA G UR RAN * & IN DA R W. RA MN ARI NE†
*Gatty Marine Laboratory, University of St Andrews yLife Sciences Department, University of the West Indies (Received 22 July 2003; initial acceptance 26 September 2003; final acceptance 30 October 2003; MS. number: 7791)
Research on learned species discrimination has focused on the consequences of early experience. However, in species where parental care is limited or absent, including most fish, juveniles have fewer opportunities to learn from adult conspecifics. We examined male mate recognition in Trinidadian guppies, Poecilia reticulata, and in their sister species, the swamp guppy, P. picta. Choice tests revealed that males from localities where their species is the only poeciliid present initially mated with conspecific and heterospecific females at random. In contrast, P. reticulata and P. picta found in sympatry preferred their own females. We then investigated the acquisition of mating discrimination by wild P. reticulata males from two allopatric populations. Males that were allowed to interact with females of both species learned within 4 days to distinguish conspecific partners, and within a week their species discrimination matched that of sympatric populations. This study confirms that learning is important in the acquisition of adult mating preferences and shows why learned mate preferences can be important in the last stages of speciation. Ó 2004 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
As the large literature on sexual imprinting in birds reveals, learned mate preferences are important in speciation (Irwin & Price 1999). Lorenz’s (1937) classic work on imprinting, which showed that hand-reared birds may preferentially court humans when adult, demonstrated the potency of early experience in shaping mate choice. Parental care, and interactions with conspecifics, provide young animals with many opportunities to learn to identify mating partners from their own species. By the same token, reproductive isolation may be impeded if animals choose, as a consequence of learning, to mate across population or species boundaries. Experiments have shown that early experience of heterospecific adults can result in preferences for mates from the wrong species (Clayton 1988; Oetting et al. 1995). Investigations in the wild have confirmed that cross-fostering leads to heterospecific pairings (Slagsvold et al. 2002). Learned mate preferences are likely to be particularly important in the last stages of speciation, because they help to determine the rate at which complete premating isolation is accomplished (Irwin & Price 1999). Sexual imprinting is just one learning mechanism that can shape the mating preferences of adult animals. Other Correspondence: A. E. Magurran, Gatty Marine Laboratory, School of Biology, University of St Andrews, St Andrews, Fife KY16 8LB, U.K. (email: [email protected]). I. W. Ramnarine is at the Department of Life Sciences, University of the West Indies, St Augustine, Trinidad and Tobago. 0003e3472/03/$30.00/0
forms of recognition learning, and their contribution to the evolution and maintenance of barriers between species however, have, been neglected. This is an important omission because parental care, which creates the environment in which sexual imprinting is possible (Grant & Grant 1997; Shettleworth 1998), is not universal. Juveniles of many fish species, for example, are left to fend for themselves. Furthermore, recognition of mates is just one learning challenge that animals face. Acquired predator recognition, and the discrimination of familiar or cooperative individuals, is well documented for juveniles and adults alike (Barber & Wright 2001; Seppa¨ et al. 2001). Cyprinid fish can even learn to recognize familiar heterospecifics (Ward et al. 2003). Irwin & Price (1999) speculated that, since the benefits of recognizing particular individuals persist throughout life, the ability to learn to distinguish the traits of others must also remain. They argued that species recognition is an extension of individual recognition. Although the importance of early experience in shaping mating preferences has been extensively investigated, few studies have explored another potentially important source of mate discrimination, learning by sexually mature individuals. The Trinidadian guppy, Poecilia reticulata, provides one of the few documented examples of this. Male guppies mate promiscuously and show preferences for unfamiliar conspecifics (Kelley et al. 1999). In a pioneering study, Haskins & Haskins (1949) presented male guppies with females of three poeciliid species
1077 Ó 2004 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
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ANIMAL BEHAVIOUR, 67, 6
females. As a result, we cannot be sure whether the observed response is a product of learning, familiarization with the experimental set-up or a reaction to some change in female behaviour over time. Finally, we have few insights into the shape of the learning curve. Because data were collected at varying intervals, Haskins & Haskins’ study does not tell us how quickly discrimination was achieved or whether it was abrupt or gradual. This information is needed to shed light on the nature of the recognition learning involved, and to understand the contribution of learned mate discrimination to the evolution of premating isolation. In this study, we began by quantifying the mating choices of male guppies of both species in sympatry and allopatry. We then confirmed that male guppies can learn to discriminate conspecific and heterospecific females upon secondary contact, and determined how quickly and how consistently this discrimination was achieved.
(P. reticulata, P. picta and P. vivipara). These male guppies, which had had no experience of heterospecifics, initially directed most of their courtship towards swamp guppy, P. picta, females. (Poecilia picta and P. reticulata are sister species that diverged approximately 5 million years ago; Breden et al. 1999; F. Breden, personal communication.) After about a week, however, the majority of sneaky matings were with conspecific females. Haskins & Haskins (1949) also reported that discrimination of the correct females occurred with a high degree of accuracy after the fish had been together for about 20 days. Assortative mating driven by female preferences for particular male phenotypes has the potential to fuel speciation in the guppy system (Endler & Houde 1995). None the less, sneaky mating behaviour results in sperm transfer, even between heterospecifics (Liley 1966, S. T. Russell, personal communication), so male mating behaviour may impede the evolution of premating isolation (Magurran 1996; Parker & Partridge 1998). Haskins & Haskins’ (1949) study suggests that learned preferences by males limit heterospecific matings when two or more poeciliid species come into secondary contact. Several studies (Breden et al. 1995; Rosenqvist & Houde 1997; Jirotkul 1999) have indicated that female mate preferences are also altered by experience, demonstrating that learned discrimination is an important phenomenon in this system. Although the Haskins & Haskins (1949) study is intriguing, it leaves some important questions unanswered. First, in line with the convention at the time, the sample size was small and data from individual males were combined. We therefore have no information on individual variation, nor are there statistical analyses of male behaviour. Second, an important control was missing: there was no comparison with males from localities where poeciliids occur sympatrically; such fish would be expected consistently to prefer conspecific to heterospecific
METHODS
Baseline Mate Choice Poecilia reticulata were collected, using a one-person seine net, from the Aripo (grid reference PS 942 777) and Tacarigua Rivers (PS 789 764), where they are the only poeciliid species present, and from Sumaria (PS 730 674), where guppies and P. picta occur in sympatry. Poecilia picta came from Sumaria and from the Blue River (PS 721 727, an allopatric locality) in the nearby Caroni Swamp, Trinidad. Collections were made in March 2002. All four localities are downstream, high-predation habitats where poeciliids co-occur with the pike cichlid Crenicichla alta and a range of other piscivores (Fig. 1). Male and female fish were captured, transported (in groups of about 50 in covered 20-litre buckets), and housed separately to ensure
1
0.75 P. reticulata male P. reticulata female 0.5 P. picta male P. picta female 0.25
0
1
2
3 School
4
5
Figure 1. The structure of five mixed poeciliid schools, showing the proportion of adult males and females by species. Total number of adult fish in each school was, respectively, 7, 42, 26, 71 and 77.
MAGURRAN & RAMNARINE: LEARNED MATE PREFERENCES
that there were no familiarity effects (Griffiths & Magurran 1997a) that might influence mating preferences (Kelley et al. 1999). Before each trial, two heterospecific size-matched females (one P. reticulata and one P. picta, both unfamiliar to the males) were gently placed into the test tank (45!30 cm and 30 cm high, with water 21 cm deep) and allowed to swim freely. Although females of the two species resemble one another, subtle differences in shape and marking meant that they could be distinguished. Two individually recognizable males from the same species and locality were then added. The presence of a second male ensured that the males were competing for mates (Evans & Magurran 1999), the natural situation in the wild (Magurran & Seghers 1994). One of these males was selected as the focal male, and his behaviour was monitored for 15 min from the moment that the first male began to show interest in a female. The number of sneaky mating attempts (gonopodial thrusts) by the focal male was recorded. Sneaky mating behaviour is similar in both species (Liley 1966). When the trial was over, all fish were replaced with new individuals. With the exception of Blue River P. picta (N ¼ 16 males), the sample size was 20. No male was observed more than once.
experienced exactly the same treatment. We did not repeat the experiment using P. picta males, partly because of time constraints, but also because we did not wish to overcollect wild fish. From these observations, we calculated the proportion of sneaky mating attempts directed towards the heterospecific (P. picta) female. All non-normal proportion data received an angular transformation (Sokal & Rohlf 1995) before analysis.
Ethical Note All guppies were housed at the freshwater laboratory at the University of the West Indies, St Augustine, Trinidad. Extreme care was taken during handling to ensure that fish were not stressed. The guppies were housed in large aerated aquaria (45!45 cm and 120 cm high; ca. 100 per tank) furnished with natural river gravel to provide cover and were fed twice daily with commercial flake food and hatched brine shrimp. No aggressive behaviour was observed. Windows in the laboratory ensured that the fish experienced the natural day/night cycle. Temperature was ambient (circa 25(C). After the experiment, the fish remained in the laboratory as breeding stock. No licences were required for the study.
Learned Preferences Trinidadian guppies were collected from two allopatric localities, Aripo and Tacarigua, and two sympatric localities, Sumaria and Beharrylal (PS 731 677), in February 2003. Given the mortality rates experienced in the wild (Reznick et al. 1996) it is likely that 2003 adults had been recruited to these populations since the previous collections. Poecilia picta females were collected from another two sites in the Caroni Swamp (PS 718 672 and PS 721 727) for use as tutor fish. On day 1, six guppy males were selected from each of the four localities (two allopatric and two sympatric). Their unique colour patterns meant that these males were individually identifiable. Each focal male was randomly paired with a companion male from his own locality, and the two males were then gently introduced to the test tank. This test tank (45!30!30 cm, with water 20 cm deep and furnished with a filter and natural river gravel) already contained two size-matched females, one of each species. As before, we avoided familiarity effects by selecting females from another locality. The 10-min trial started as soon as the focal male began to pursue a female. We recorded the number of sneaky mating attempts, and the identity of the female to which they were directed. At the end of the trial, the six focal males from each locality were housed in new home aquaria with the same dimensions and furnishings as the test tank, each containing three conspecific and three heterospecific females, all of them unfamiliar to the males. The same males were then tested daily for a further 5 days before being returned to their home aquarium. Focal males were paired with a different companion male during each retest and encountered different females on each occasion. The procedure was done three times (N ¼ 18 fish per locality). Data were not collected on companion males, even though they
RESULTS
Baseline Mate Choice Males of both species attempted matings with heterospecific females. None of the allopatric populations of either species showed any preference for conspecifics (Fig. 2, Table 1). However, when the species were naturally in sympatry, both P. reticulata and P. picta significantly preferred to copulate with their own females (Fig. 2, and Table 1).
Learned Preferences Repeated measures ANOVA showed no significant differences in the frequency of sneaky mating attempts between localities (F3;68 ¼ 1:752, P ¼ 0:164) or over time (F5;340 ¼ 2:091, P ¼ 0:066). There was, none the less, a significant interaction between time and allopatry/ sympatry (repeated measures ANOVA on proportion of mating attempts directed towards P. picta females: F5;345 ¼ 21:30, P!0:001; Fig. 3). BonferronieDunn post hoc tests confirmed that allopatric populations (Aripo and Tacarigua) showed a significant change in preference (P!0:001), in favour of the P. reticulata female, from day 4. In comparison, no significant pairwise comparisons between days were detected for the sympatric (Sumaria and Beharrylal) populations. We also detected a preference for P. picta females by allopatric males on day 2 (Fig. 3). DISCUSSION Our investigation shows that the ability of P. reticulata and P. picta males to identify a mating partner of the correct
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ANIMAL BEHAVIOUR, 67, 6
1
0.8
0.6
0.4
0.2
0
Aripo
Tacarigua
Sumaria (r)
Sumaria (p)
Blue River
Figure 2. Proportion (mean G 95% confidence limit) of sneaky mating attempts directed towards the P. picta female by the focal male (2002). Poecilia reticulata exists in allopatry in Aripo and Tacarigua. Blue River is an allopatric site for P. picta. Both species occur at Sumaria. Sumaria (r): P. reticulata; Sumaria (p): P. picta. Dashed line indicates 50% (random choice). N ¼ 20 males per category except Blue River (N ¼ 16).
species is determined by the composition of the ecological community in which they occur in the wild. Guppies from allopatric localities mate at random; those in sympatry do not. The consistency of the result across rivers and over time strengthens this conclusion. The results further show that male Trinidadian guppies with no experience of P. picta can learn to discriminate heterospecific and conspecific females within a few days. The transition is such that, within a week, the mating preferences of Trinidadian guppies resemble those of males from populations that naturally coexist with P. picta. The timing is consistent with work by Liley (1966), who showed that a 2.5-day exposure to unreceptive conspecific and heterospecific females was insufficient for discrimination learning in male P. reticulata. Haskins & Haskins’ (1949) study also points to the emergence of discrimination 2e5 days after the first encounter with heterospecific females. The behaviour of the males in this study from the sympatric localities was consistent over time, so we can exclude the possibility that males were habituating to the experimenTable 1. The number of sneaky mating attempts (gonopodial thrusts) directed towards the P. picta female Origin and species of focal male Allopatry Aripo (P. reticulata) Tacarigua (P. reticulata) Blue River (P. picta) Sympatry Sumaria (P. picta) Sumaria (P. reticulata)
XGSE number of sneaky mating attempts*
t19†
P
8.6G0.98 7G0.96 3.4G0.51
0.185 0.738 1.18
0.86 0.47 0.26
7.9G0.93 6.9G1.02
24.14 !0.001 6.64‡ !0.001
*Attempts per 15 min. †One-sample t tests. All data were first checked for normality using a KolmogoroveSmirnov test. ‡Non-normal data received an angular transformation and were tested against the transformed value of 0.5 (0.45).
tal set-up or responding to changes in female behaviour. Parallel responses in the replicate populations imply that the result may be generally applicable, at least to males from downstream high-predation populations. Furthermore, the observation that the same baseline choice behaviour is apparent in both poeciliids suggests that the result can be generalized beyond a single species of guppy. The observation that the origin of wild-caught fish determines initial preferences for mates might appear to represent a case of reinforcement (stronger mate discrimination in sympatry than allopatry). This, of course, would be a spurious conclusion, because the two sets of fish have had different opportunities to learn. A more general point is that this problem may afflict all studies of animals that can learn in the wild, compared with those reared and tested naı¨vely in the laboratory (Coyne & Orr 2004). Like Haskins & Haskins (1949), we found that P. picta females were preferred by naı¨ve males in the early stages of the experiment. Haskins & Haskins observed that P. picta females cease swimming when pursued by male guppies, a response that resembles receptive behaviour in female guppies, and this reaction, which we also noted, probably explains the initial heterospecific preference. Guppies recognize and associate with familiar individuals in the laboratory and in the wild (Griffiths & Magurran 1997b, 1998). Familiarity recognition in this species emerges at around 12 days (Griffiths & Magurran 1997a). If male discrimination of conspecific partners were a form of familiarity learning, we would expect it to become established over a similar timescale. Our fish encountered different female fish in each test, so the response cannot have been based on familiarity with particular individuals but rather on learned recognition of the chemical and visual cues that distinguish the two types of female. The mate identification that we observed is more consistent with individual recognition, as Irwin & Price (1999) proposed. Research on inspection behaviour in guppies and sticklebacks, Gasterosteus aculeatus, indicates that learned individual recognition may be possible
MAGURRAN & RAMNARINE: LEARNED MATE PREFERENCES
Beharrylal Sumaria
0.8
Aripo Tacarigua
0.6
0.4
0.2
0
1
2
3
4
5
6
Day
Figure 3. Proportion (mean G 95% confidence limit) of sneaky mating attempts directed towards the P. picta female by the focal guppy male over successive days of the experiment (2003). Dashed line represents 50% (random choice). N ¼ 18 males per population.
after a few hours (Milinski et al. 1990; Dugatkin & Alfieri 1991) although these results may also be attributable to condition-dependent recognition (see discussion in Griffiths & Magurran 1997a). Liley (1966) established that it was not the actual copulation, but rather the events leading up to it, that mediates discrimination learning. He also found that P. reticulata males reared with P. picta females initially courted the heterospecific females from the point of first gonopodial development, but they soon began to display and court females of their own species. This experiment and others in which males were separated from females for varying periods led Liley (1966) to propose that there may be a sensitive period during development when P. reticulata males learn to discriminate males and females and possibly even to distinguish between females. Similarly, exposure to certain male phenotypes influences the mating choices of adult female guppies (Breden et al. 1995; Rosenqvist & Houde 1997; Jirotkul, 1999). Early experience can thus shape mating preferences in guppies. However, Shettleworth (1998) stressed that the acquisition of sexual preferences is not a single process. Many bird species show a predisposition to recognize conspecific mates (Immelmann 1972). These preferences are moulded by early events, including filial imprinting, but are consolidated by sexual contact later in life (Bischof 1994). The guppy system clearly demonstrates that learned mate discrimination continues throughout life. Furthermore, this species provides evidence that learned discrimination of species is underpinned by the same mechanisms that produce learned discrimination of conspecifics (Ryan & Rand 1993). Spalding (1873, cited in Irwin & Price 1999) first pointed out that learned recognition can, through selection, result in the evolution of innate recognition mechanisms (Fear & Price 1998). One process by which learned preferences are translated into innate ones is known as assimilation (Waddington 1959; Fear & Price 1998). Our
experiment contrasted the behaviour of guppies from allopatric localities with those that occur sympatrically with P. picta and thus have already had ample opportunity to acquire species discrimination. The next step is to determine whether innate recognition mechanisms are stronger or whether learning is accomplished more readily in populations found in sympatry. Acknowledgments We thank Rajindra Mahabir and Stephen Russell for help with fish collection and maintenance, Jerry Coyne and our referees for their helpful comments and the Royal Society and Natural Environment Research Council, U.K., for financial support.
References Barber, I. & Wright, H. A. 2001. How strong are familiarity preferences in shoaling fish? Animal Behaviour, 61, 975e979. Bischof, H.-J. 1994. Sexual imprinting as a two-stage process. In: Causal Mechanisms of Behavioural Development (Ed. by J. A. Hogan & J. J. Bolhuis), pp. 82e97. Cambridge: Cambridge University Press. Breden, F., Novinger, D. & Schubert, A. 1995. The effect of experience on mate choice in the Trinidad guppy, Poecilia reticulata. Environmental Biology of Fishes, 7, 323e328. Breden, F., Ptacek, M. B., Rashed, M., Taphorn, D. & Figueiredo, C. A. 1999. Molecular phylogeny of the live-bearing fish genus Poecilia (Cyprinodontiformes: Poeciliidae). Molecular Phylogenetics and Evolution, 12, 95e104. Clayton, N. S. 1988. Song learning and mate choice in estrilidid finches raised by two species. Animal Behaviour, 36, 1589e1600. Coyne, J. A. & Orr, H. A. 2004. Speciation. Sunderland, Massachusetts: Sinauer. Dugatkin, L. A. & Alfieri, M. 1991. Guppies and the tit for tat strategy: preference based on past interaction. Behavioral Ecology and Sociobiology, 28, 243e246.
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Endler, J. A. & Houde, A. E. 1995. Geographic variation in female preferences for male traits in Poecilia reticulata. Evolution, 49, 456e468. Evans, J. P. & Magurran, A. E. 1999. Male mating behaviour and sperm production characteristics under varying sperm competition risk in guppies. Animal Behaviour, 58, 1101e1106. Fear, K. K. & Price, T. 1998. The adaptive surface in ecology. Oikos, 82, 440e448. Grant, P. R. & Grant, B. R. 1997. Hybridization, sexual imprinting and mate choice. American Naturalist, 149, 1e28. Griffiths, S. W. & Magurran, A. E. 1997a. Familiarity in schooling fish: how long does it take to acquire? Animal Behaviour, 53, 945e949. Griffiths, S. W. & Magurran, A. E. 1997b. Schooling preferences for familiar fish vary with group size in a wild guppy population. Proceedings of the Royal Society of London, Series B, 264, 547e551. Griffiths, S. W. & Magurran, A. E. 1998. Sex and schooling behaviour in Trinidadian guppies. Animal Behaviour, 56, 689e693. Haskins, C. P. & Haskins, E. F. 1949. The role of sexual selection as an isolating mechanism in three species of poeciliid fishes. Evolution, 3, 160e169. Immelmann, K. 1972. Sexual and other longterm aspects of imprinting in birds and other species. Advances in the Study of Behavior, 4, 147e174. Irwin, D. E. & Price, T. 1999. Sexual imprinting, learning and speciation. Heredity, 82, 347e354. Jirotkul, M. 1999. Operational sex ratio influences female preferences and maleemale competition in guppies. Animal Behaviour, 58, 287e294. Kelley, J. L., Graves, J. A. & Magurran, A. E. 1999. Familiarity breeds contempt in guppies. Nature, 401, 661e662. Liley, N. R. 1966. Ethological isolating mechanisms in four sympatric species of Poeciliid fishes. Behaviour Supplement, 13, 1e197. Lorenz, K. 1937. The companion in the bird’s world. Auk, 54, 245e273. Magurran, A. E. 1996. Battle of the sexes. Nature, 383, 307. Magurran, A. E. & Seghers, B. H. 1994. Sexual conflict as a consequence of ecology: evidence from guppy, Poecilia reticulata,
populations in Trinidad. Proceedings of the Royal Society of London, Series B, 255, 31e36. ¨ lling, D. & Kettler, R. 1990. Tit for tat: stickleMilinski, M., Ku backs ‘trusting’ a cooperating partner. Behavioral Ecology, 1, 7e11. ¨ ve, E. & Bischof, H. J. 1995. Sexual imprinting as Oetting, S., Pro a two-stage process: mechanisms of information storage and stabilization. Animal Behaviour, 50, 393e403. Parker, G. A. & Partridge, L. 1998. Sexual conflict and speciation. Philosophical Transactions of the Royal Society of London, Series B, 353, 261e274. Reznick, D. N., Butler, M. J., Rodd, F. H. & Ross, P. 1996. Lifehistory evolution in guppies (Poecilia reticulata). 6. Differential mortality as a mechanism for natural selection. Evolution, 50, 1651e1660. Rosenqvist, G. & Houde, A. 1997. Prior exposure to male phenotypes influences mate choice in the guppy, Poecilia reticulata. Behavioral Ecology, 8, 194e198. Ryan, M. J. & Rand, A. S. 1993. Species recognition and sexual selection as a unitary problem in animal communication. Evolution, 47, 647e657. Seppa¨, T., Laurila, A., Peuhkuri, N., Pironen, J. & Lower, N. 2001. Early familiarity has fitness consequences for Arctic char (Salvelinus alpinus) juveniles. Canadian Journal of Fisheries and Aquatic Sciences, 58, 1380e1385. Shettleworth, S. J. 1998. Cognition, Evolution and Behavior. New York: Oxford University Press. Slagsvold, T., Hansen, B. T., Johannessen, L. E. & Lifjeld, J. T. 2002. Mate choice and imprinting in bird studies by crossfostering in the wild. Proceedings of the Royal Society of London, Series B, 269, 1449e1455. Sokal, R. R. & Rohlf, F. J. 1995. Biometry. New York: W. H. Freeman. Waddington, C. H. 1959. Canalization of development and genetic assimilation of acquired characters. Nature, 183, 1654e1655. Ward, A. J. W., Axford, S. & Krause, J. 2003. Cross-species familiarity in shoaling fishes. Proceedings of the Royal Society of London, Series B, 270, 1157e1161.
Learned mate recognition and reproductive isolation in guppies AN NE E. MA G UR RAN * & IN DA R W. RA MN ARI NE†
*Gatty Marine Laboratory, University of St Andrews yLife Sciences Department, University of the West Indies (Received 22 July 2003; initial acceptance 26 September 2003; final acceptance 30 October 2003; MS. number: 7791)
Research on learned species discrimination has focused on the consequences of early experience. However, in species where parental care is limited or absent, including most fish, juveniles have fewer opportunities to learn from adult conspecifics. We examined male mate recognition in Trinidadian guppies, Poecilia reticulata, and in their sister species, the swamp guppy, P. picta. Choice tests revealed that males from localities where their species is the only poeciliid present initially mated with conspecific and heterospecific females at random. In contrast, P. reticulata and P. picta found in sympatry preferred their own females. We then investigated the acquisition of mating discrimination by wild P. reticulata males from two allopatric populations. Males that were allowed to interact with females of both species learned within 4 days to distinguish conspecific partners, and within a week their species discrimination matched that of sympatric populations. This study confirms that learning is important in the acquisition of adult mating preferences and shows why learned mate preferences can be important in the last stages of speciation. Ó 2004 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
As the large literature on sexual imprinting in birds reveals, learned mate preferences are important in speciation (Irwin & Price 1999). Lorenz’s (1937) classic work on imprinting, which showed that hand-reared birds may preferentially court humans when adult, demonstrated the potency of early experience in shaping mate choice. Parental care, and interactions with conspecifics, provide young animals with many opportunities to learn to identify mating partners from their own species. By the same token, reproductive isolation may be impeded if animals choose, as a consequence of learning, to mate across population or species boundaries. Experiments have shown that early experience of heterospecific adults can result in preferences for mates from the wrong species (Clayton 1988; Oetting et al. 1995). Investigations in the wild have confirmed that cross-fostering leads to heterospecific pairings (Slagsvold et al. 2002). Learned mate preferences are likely to be particularly important in the last stages of speciation, because they help to determine the rate at which complete premating isolation is accomplished (Irwin & Price 1999). Sexual imprinting is just one learning mechanism that can shape the mating preferences of adult animals. Other Correspondence: A. E. Magurran, Gatty Marine Laboratory, School of Biology, University of St Andrews, St Andrews, Fife KY16 8LB, U.K. (email: [email protected]). I. W. Ramnarine is at the Department of Life Sciences, University of the West Indies, St Augustine, Trinidad and Tobago. 0003e3472/03/$30.00/0
forms of recognition learning, and their contribution to the evolution and maintenance of barriers between species however, have, been neglected. This is an important omission because parental care, which creates the environment in which sexual imprinting is possible (Grant & Grant 1997; Shettleworth 1998), is not universal. Juveniles of many fish species, for example, are left to fend for themselves. Furthermore, recognition of mates is just one learning challenge that animals face. Acquired predator recognition, and the discrimination of familiar or cooperative individuals, is well documented for juveniles and adults alike (Barber & Wright 2001; Seppa¨ et al. 2001). Cyprinid fish can even learn to recognize familiar heterospecifics (Ward et al. 2003). Irwin & Price (1999) speculated that, since the benefits of recognizing particular individuals persist throughout life, the ability to learn to distinguish the traits of others must also remain. They argued that species recognition is an extension of individual recognition. Although the importance of early experience in shaping mating preferences has been extensively investigated, few studies have explored another potentially important source of mate discrimination, learning by sexually mature individuals. The Trinidadian guppy, Poecilia reticulata, provides one of the few documented examples of this. Male guppies mate promiscuously and show preferences for unfamiliar conspecifics (Kelley et al. 1999). In a pioneering study, Haskins & Haskins (1949) presented male guppies with females of three poeciliid species
1077 Ó 2004 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
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ANIMAL BEHAVIOUR, 67, 6
females. As a result, we cannot be sure whether the observed response is a product of learning, familiarization with the experimental set-up or a reaction to some change in female behaviour over time. Finally, we have few insights into the shape of the learning curve. Because data were collected at varying intervals, Haskins & Haskins’ study does not tell us how quickly discrimination was achieved or whether it was abrupt or gradual. This information is needed to shed light on the nature of the recognition learning involved, and to understand the contribution of learned mate discrimination to the evolution of premating isolation. In this study, we began by quantifying the mating choices of male guppies of both species in sympatry and allopatry. We then confirmed that male guppies can learn to discriminate conspecific and heterospecific females upon secondary contact, and determined how quickly and how consistently this discrimination was achieved.
(P. reticulata, P. picta and P. vivipara). These male guppies, which had had no experience of heterospecifics, initially directed most of their courtship towards swamp guppy, P. picta, females. (Poecilia picta and P. reticulata are sister species that diverged approximately 5 million years ago; Breden et al. 1999; F. Breden, personal communication.) After about a week, however, the majority of sneaky matings were with conspecific females. Haskins & Haskins (1949) also reported that discrimination of the correct females occurred with a high degree of accuracy after the fish had been together for about 20 days. Assortative mating driven by female preferences for particular male phenotypes has the potential to fuel speciation in the guppy system (Endler & Houde 1995). None the less, sneaky mating behaviour results in sperm transfer, even between heterospecifics (Liley 1966, S. T. Russell, personal communication), so male mating behaviour may impede the evolution of premating isolation (Magurran 1996; Parker & Partridge 1998). Haskins & Haskins’ (1949) study suggests that learned preferences by males limit heterospecific matings when two or more poeciliid species come into secondary contact. Several studies (Breden et al. 1995; Rosenqvist & Houde 1997; Jirotkul 1999) have indicated that female mate preferences are also altered by experience, demonstrating that learned discrimination is an important phenomenon in this system. Although the Haskins & Haskins (1949) study is intriguing, it leaves some important questions unanswered. First, in line with the convention at the time, the sample size was small and data from individual males were combined. We therefore have no information on individual variation, nor are there statistical analyses of male behaviour. Second, an important control was missing: there was no comparison with males from localities where poeciliids occur sympatrically; such fish would be expected consistently to prefer conspecific to heterospecific
METHODS
Baseline Mate Choice Poecilia reticulata were collected, using a one-person seine net, from the Aripo (grid reference PS 942 777) and Tacarigua Rivers (PS 789 764), where they are the only poeciliid species present, and from Sumaria (PS 730 674), where guppies and P. picta occur in sympatry. Poecilia picta came from Sumaria and from the Blue River (PS 721 727, an allopatric locality) in the nearby Caroni Swamp, Trinidad. Collections were made in March 2002. All four localities are downstream, high-predation habitats where poeciliids co-occur with the pike cichlid Crenicichla alta and a range of other piscivores (Fig. 1). Male and female fish were captured, transported (in groups of about 50 in covered 20-litre buckets), and housed separately to ensure
1
0.75 P. reticulata male P. reticulata female 0.5 P. picta male P. picta female 0.25
0
1
2
3 School
4
5
Figure 1. The structure of five mixed poeciliid schools, showing the proportion of adult males and females by species. Total number of adult fish in each school was, respectively, 7, 42, 26, 71 and 77.
MAGURRAN & RAMNARINE: LEARNED MATE PREFERENCES
that there were no familiarity effects (Griffiths & Magurran 1997a) that might influence mating preferences (Kelley et al. 1999). Before each trial, two heterospecific size-matched females (one P. reticulata and one P. picta, both unfamiliar to the males) were gently placed into the test tank (45!30 cm and 30 cm high, with water 21 cm deep) and allowed to swim freely. Although females of the two species resemble one another, subtle differences in shape and marking meant that they could be distinguished. Two individually recognizable males from the same species and locality were then added. The presence of a second male ensured that the males were competing for mates (Evans & Magurran 1999), the natural situation in the wild (Magurran & Seghers 1994). One of these males was selected as the focal male, and his behaviour was monitored for 15 min from the moment that the first male began to show interest in a female. The number of sneaky mating attempts (gonopodial thrusts) by the focal male was recorded. Sneaky mating behaviour is similar in both species (Liley 1966). When the trial was over, all fish were replaced with new individuals. With the exception of Blue River P. picta (N ¼ 16 males), the sample size was 20. No male was observed more than once.
experienced exactly the same treatment. We did not repeat the experiment using P. picta males, partly because of time constraints, but also because we did not wish to overcollect wild fish. From these observations, we calculated the proportion of sneaky mating attempts directed towards the heterospecific (P. picta) female. All non-normal proportion data received an angular transformation (Sokal & Rohlf 1995) before analysis.
Ethical Note All guppies were housed at the freshwater laboratory at the University of the West Indies, St Augustine, Trinidad. Extreme care was taken during handling to ensure that fish were not stressed. The guppies were housed in large aerated aquaria (45!45 cm and 120 cm high; ca. 100 per tank) furnished with natural river gravel to provide cover and were fed twice daily with commercial flake food and hatched brine shrimp. No aggressive behaviour was observed. Windows in the laboratory ensured that the fish experienced the natural day/night cycle. Temperature was ambient (circa 25(C). After the experiment, the fish remained in the laboratory as breeding stock. No licences were required for the study.
Learned Preferences Trinidadian guppies were collected from two allopatric localities, Aripo and Tacarigua, and two sympatric localities, Sumaria and Beharrylal (PS 731 677), in February 2003. Given the mortality rates experienced in the wild (Reznick et al. 1996) it is likely that 2003 adults had been recruited to these populations since the previous collections. Poecilia picta females were collected from another two sites in the Caroni Swamp (PS 718 672 and PS 721 727) for use as tutor fish. On day 1, six guppy males were selected from each of the four localities (two allopatric and two sympatric). Their unique colour patterns meant that these males were individually identifiable. Each focal male was randomly paired with a companion male from his own locality, and the two males were then gently introduced to the test tank. This test tank (45!30!30 cm, with water 20 cm deep and furnished with a filter and natural river gravel) already contained two size-matched females, one of each species. As before, we avoided familiarity effects by selecting females from another locality. The 10-min trial started as soon as the focal male began to pursue a female. We recorded the number of sneaky mating attempts, and the identity of the female to which they were directed. At the end of the trial, the six focal males from each locality were housed in new home aquaria with the same dimensions and furnishings as the test tank, each containing three conspecific and three heterospecific females, all of them unfamiliar to the males. The same males were then tested daily for a further 5 days before being returned to their home aquarium. Focal males were paired with a different companion male during each retest and encountered different females on each occasion. The procedure was done three times (N ¼ 18 fish per locality). Data were not collected on companion males, even though they
RESULTS
Baseline Mate Choice Males of both species attempted matings with heterospecific females. None of the allopatric populations of either species showed any preference for conspecifics (Fig. 2, Table 1). However, when the species were naturally in sympatry, both P. reticulata and P. picta significantly preferred to copulate with their own females (Fig. 2, and Table 1).
Learned Preferences Repeated measures ANOVA showed no significant differences in the frequency of sneaky mating attempts between localities (F3;68 ¼ 1:752, P ¼ 0:164) or over time (F5;340 ¼ 2:091, P ¼ 0:066). There was, none the less, a significant interaction between time and allopatry/ sympatry (repeated measures ANOVA on proportion of mating attempts directed towards P. picta females: F5;345 ¼ 21:30, P!0:001; Fig. 3). BonferronieDunn post hoc tests confirmed that allopatric populations (Aripo and Tacarigua) showed a significant change in preference (P!0:001), in favour of the P. reticulata female, from day 4. In comparison, no significant pairwise comparisons between days were detected for the sympatric (Sumaria and Beharrylal) populations. We also detected a preference for P. picta females by allopatric males on day 2 (Fig. 3). DISCUSSION Our investigation shows that the ability of P. reticulata and P. picta males to identify a mating partner of the correct
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ANIMAL BEHAVIOUR, 67, 6
1
0.8
0.6
0.4
0.2
0
Aripo
Tacarigua
Sumaria (r)
Sumaria (p)
Blue River
Figure 2. Proportion (mean G 95% confidence limit) of sneaky mating attempts directed towards the P. picta female by the focal male (2002). Poecilia reticulata exists in allopatry in Aripo and Tacarigua. Blue River is an allopatric site for P. picta. Both species occur at Sumaria. Sumaria (r): P. reticulata; Sumaria (p): P. picta. Dashed line indicates 50% (random choice). N ¼ 20 males per category except Blue River (N ¼ 16).
species is determined by the composition of the ecological community in which they occur in the wild. Guppies from allopatric localities mate at random; those in sympatry do not. The consistency of the result across rivers and over time strengthens this conclusion. The results further show that male Trinidadian guppies with no experience of P. picta can learn to discriminate heterospecific and conspecific females within a few days. The transition is such that, within a week, the mating preferences of Trinidadian guppies resemble those of males from populations that naturally coexist with P. picta. The timing is consistent with work by Liley (1966), who showed that a 2.5-day exposure to unreceptive conspecific and heterospecific females was insufficient for discrimination learning in male P. reticulata. Haskins & Haskins’ (1949) study also points to the emergence of discrimination 2e5 days after the first encounter with heterospecific females. The behaviour of the males in this study from the sympatric localities was consistent over time, so we can exclude the possibility that males were habituating to the experimenTable 1. The number of sneaky mating attempts (gonopodial thrusts) directed towards the P. picta female Origin and species of focal male Allopatry Aripo (P. reticulata) Tacarigua (P. reticulata) Blue River (P. picta) Sympatry Sumaria (P. picta) Sumaria (P. reticulata)
XGSE number of sneaky mating attempts*
t19†
P
8.6G0.98 7G0.96 3.4G0.51
0.185 0.738 1.18
0.86 0.47 0.26
7.9G0.93 6.9G1.02
24.14 !0.001 6.64‡ !0.001
*Attempts per 15 min. †One-sample t tests. All data were first checked for normality using a KolmogoroveSmirnov test. ‡Non-normal data received an angular transformation and were tested against the transformed value of 0.5 (0.45).
tal set-up or responding to changes in female behaviour. Parallel responses in the replicate populations imply that the result may be generally applicable, at least to males from downstream high-predation populations. Furthermore, the observation that the same baseline choice behaviour is apparent in both poeciliids suggests that the result can be generalized beyond a single species of guppy. The observation that the origin of wild-caught fish determines initial preferences for mates might appear to represent a case of reinforcement (stronger mate discrimination in sympatry than allopatry). This, of course, would be a spurious conclusion, because the two sets of fish have had different opportunities to learn. A more general point is that this problem may afflict all studies of animals that can learn in the wild, compared with those reared and tested naı¨vely in the laboratory (Coyne & Orr 2004). Like Haskins & Haskins (1949), we found that P. picta females were preferred by naı¨ve males in the early stages of the experiment. Haskins & Haskins observed that P. picta females cease swimming when pursued by male guppies, a response that resembles receptive behaviour in female guppies, and this reaction, which we also noted, probably explains the initial heterospecific preference. Guppies recognize and associate with familiar individuals in the laboratory and in the wild (Griffiths & Magurran 1997b, 1998). Familiarity recognition in this species emerges at around 12 days (Griffiths & Magurran 1997a). If male discrimination of conspecific partners were a form of familiarity learning, we would expect it to become established over a similar timescale. Our fish encountered different female fish in each test, so the response cannot have been based on familiarity with particular individuals but rather on learned recognition of the chemical and visual cues that distinguish the two types of female. The mate identification that we observed is more consistent with individual recognition, as Irwin & Price (1999) proposed. Research on inspection behaviour in guppies and sticklebacks, Gasterosteus aculeatus, indicates that learned individual recognition may be possible
MAGURRAN & RAMNARINE: LEARNED MATE PREFERENCES
Beharrylal Sumaria
0.8
Aripo Tacarigua
0.6
0.4
0.2
0
1
2
3
4
5
6
Day
Figure 3. Proportion (mean G 95% confidence limit) of sneaky mating attempts directed towards the P. picta female by the focal guppy male over successive days of the experiment (2003). Dashed line represents 50% (random choice). N ¼ 18 males per population.
after a few hours (Milinski et al. 1990; Dugatkin & Alfieri 1991) although these results may also be attributable to condition-dependent recognition (see discussion in Griffiths & Magurran 1997a). Liley (1966) established that it was not the actual copulation, but rather the events leading up to it, that mediates discrimination learning. He also found that P. reticulata males reared with P. picta females initially courted the heterospecific females from the point of first gonopodial development, but they soon began to display and court females of their own species. This experiment and others in which males were separated from females for varying periods led Liley (1966) to propose that there may be a sensitive period during development when P. reticulata males learn to discriminate males and females and possibly even to distinguish between females. Similarly, exposure to certain male phenotypes influences the mating choices of adult female guppies (Breden et al. 1995; Rosenqvist & Houde 1997; Jirotkul, 1999). Early experience can thus shape mating preferences in guppies. However, Shettleworth (1998) stressed that the acquisition of sexual preferences is not a single process. Many bird species show a predisposition to recognize conspecific mates (Immelmann 1972). These preferences are moulded by early events, including filial imprinting, but are consolidated by sexual contact later in life (Bischof 1994). The guppy system clearly demonstrates that learned mate discrimination continues throughout life. Furthermore, this species provides evidence that learned discrimination of species is underpinned by the same mechanisms that produce learned discrimination of conspecifics (Ryan & Rand 1993). Spalding (1873, cited in Irwin & Price 1999) first pointed out that learned recognition can, through selection, result in the evolution of innate recognition mechanisms (Fear & Price 1998). One process by which learned preferences are translated into innate ones is known as assimilation (Waddington 1959; Fear & Price 1998). Our
experiment contrasted the behaviour of guppies from allopatric localities with those that occur sympatrically with P. picta and thus have already had ample opportunity to acquire species discrimination. The next step is to determine whether innate recognition mechanisms are stronger or whether learning is accomplished more readily in populations found in sympatry. Acknowledgments We thank Rajindra Mahabir and Stephen Russell for help with fish collection and maintenance, Jerry Coyne and our referees for their helpful comments and the Royal Society and Natural Environment Research Council, U.K., for financial support.
References Barber, I. & Wright, H. A. 2001. How strong are familiarity preferences in shoaling fish? Animal Behaviour, 61, 975e979. Bischof, H.-J. 1994. Sexual imprinting as a two-stage process. In: Causal Mechanisms of Behavioural Development (Ed. by J. A. Hogan & J. J. Bolhuis), pp. 82e97. Cambridge: Cambridge University Press. Breden, F., Novinger, D. & Schubert, A. 1995. The effect of experience on mate choice in the Trinidad guppy, Poecilia reticulata. Environmental Biology of Fishes, 7, 323e328. Breden, F., Ptacek, M. B., Rashed, M., Taphorn, D. & Figueiredo, C. A. 1999. Molecular phylogeny of the live-bearing fish genus Poecilia (Cyprinodontiformes: Poeciliidae). Molecular Phylogenetics and Evolution, 12, 95e104. Clayton, N. S. 1988. Song learning and mate choice in estrilidid finches raised by two species. Animal Behaviour, 36, 1589e1600. Coyne, J. A. & Orr, H. A. 2004. Speciation. Sunderland, Massachusetts: Sinauer. Dugatkin, L. A. & Alfieri, M. 1991. Guppies and the tit for tat strategy: preference based on past interaction. Behavioral Ecology and Sociobiology, 28, 243e246.
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Endler, J. A. & Houde, A. E. 1995. Geographic variation in female preferences for male traits in Poecilia reticulata. Evolution, 49, 456e468. Evans, J. P. & Magurran, A. E. 1999. Male mating behaviour and sperm production characteristics under varying sperm competition risk in guppies. Animal Behaviour, 58, 1101e1106. Fear, K. K. & Price, T. 1998. The adaptive surface in ecology. Oikos, 82, 440e448. Grant, P. R. & Grant, B. R. 1997. Hybridization, sexual imprinting and mate choice. American Naturalist, 149, 1e28. Griffiths, S. W. & Magurran, A. E. 1997a. Familiarity in schooling fish: how long does it take to acquire? Animal Behaviour, 53, 945e949. Griffiths, S. W. & Magurran, A. E. 1997b. Schooling preferences for familiar fish vary with group size in a wild guppy population. Proceedings of the Royal Society of London, Series B, 264, 547e551. Griffiths, S. W. & Magurran, A. E. 1998. Sex and schooling behaviour in Trinidadian guppies. Animal Behaviour, 56, 689e693. Haskins, C. P. & Haskins, E. F. 1949. The role of sexual selection as an isolating mechanism in three species of poeciliid fishes. Evolution, 3, 160e169. Immelmann, K. 1972. Sexual and other longterm aspects of imprinting in birds and other species. Advances in the Study of Behavior, 4, 147e174. Irwin, D. E. & Price, T. 1999. Sexual imprinting, learning and speciation. Heredity, 82, 347e354. Jirotkul, M. 1999. Operational sex ratio influences female preferences and maleemale competition in guppies. Animal Behaviour, 58, 287e294. Kelley, J. L., Graves, J. A. & Magurran, A. E. 1999. Familiarity breeds contempt in guppies. Nature, 401, 661e662. Liley, N. R. 1966. Ethological isolating mechanisms in four sympatric species of Poeciliid fishes. Behaviour Supplement, 13, 1e197. Lorenz, K. 1937. The companion in the bird’s world. Auk, 54, 245e273. Magurran, A. E. 1996. Battle of the sexes. Nature, 383, 307. Magurran, A. E. & Seghers, B. H. 1994. Sexual conflict as a consequence of ecology: evidence from guppy, Poecilia reticulata,
populations in Trinidad. Proceedings of the Royal Society of London, Series B, 255, 31e36. ¨ lling, D. & Kettler, R. 1990. Tit for tat: stickleMilinski, M., Ku backs ‘trusting’ a cooperating partner. Behavioral Ecology, 1, 7e11. ¨ ve, E. & Bischof, H. J. 1995. Sexual imprinting as Oetting, S., Pro a two-stage process: mechanisms of information storage and stabilization. Animal Behaviour, 50, 393e403. Parker, G. A. & Partridge, L. 1998. Sexual conflict and speciation. Philosophical Transactions of the Royal Society of London, Series B, 353, 261e274. Reznick, D. N., Butler, M. J., Rodd, F. H. & Ross, P. 1996. Lifehistory evolution in guppies (Poecilia reticulata). 6. Differential mortality as a mechanism for natural selection. Evolution, 50, 1651e1660. Rosenqvist, G. & Houde, A. 1997. Prior exposure to male phenotypes influences mate choice in the guppy, Poecilia reticulata. Behavioral Ecology, 8, 194e198. Ryan, M. J. & Rand, A. S. 1993. Species recognition and sexual selection as a unitary problem in animal communication. Evolution, 47, 647e657. Seppa¨, T., Laurila, A., Peuhkuri, N., Pironen, J. & Lower, N. 2001. Early familiarity has fitness consequences for Arctic char (Salvelinus alpinus) juveniles. Canadian Journal of Fisheries and Aquatic Sciences, 58, 1380e1385. Shettleworth, S. J. 1998. Cognition, Evolution and Behavior. New York: Oxford University Press. Slagsvold, T., Hansen, B. T., Johannessen, L. E. & Lifjeld, J. T. 2002. Mate choice and imprinting in bird studies by crossfostering in the wild. Proceedings of the Royal Society of London, Series B, 269, 1449e1455. Sokal, R. R. & Rohlf, F. J. 1995. Biometry. New York: W. H. Freeman. Waddington, C. H. 1959. Canalization of development and genetic assimilation of acquired characters. Nature, 183, 1654e1655. Ward, A. J. W., Axford, S. & Krause, J. 2003. Cross-species familiarity in shoaling fishes. Proceedings of the Royal Society of London, Series B, 270, 1157e1161.