Female associative behavior accompanies morphological distinction in two Panamanian populations of the molly Poecilia gilli (Kner) Rebecca C. Jordana, David V. Howe, Amanda Beaversb, Angela Deana, and James L. Gouldc Department of Ecology, Evolution, and Natural Resources Rutgers, the State University of New Jersey New Brunswick, New Jersey 08901 USA ABSTRACT We investigated the associative behavior of the female molly (Pisces: Poeciliidae), Poecilia gilli (Kner), collected from two Panamanian populations—one from a clear running forest stream and the other from an impacted suburban stream. We tested the hypotheses that females would preferentially associate with males and females of their local population versus the foreign population and that males would prefer local females. We also tested the hypothesis that morphological distinctions between the two populations can be made. We found that females preferentially associated with local males but preferred neither local nor foreign females. Males exhibited no preference for females based on stream of origin. Dorsal fin height differed between populations but sex had no effect on this trait. Ecological factors or female preference could be responsible for this divergence, which could be reinforced by female preference for local males. INTRODUCTION The mollies (genus Poecilia, group Mollienesia) are poeciliid fishes found widely throughout the new world (Breden et al. 1999). These diverse fishes provide an interesting opportunity to investigate population dynamics in light of sexual selection. Males exhibit variation in mating behavior and presumed sexually selected traits among and within both species and populations (e.g., Balsano et al. 1985, Farr 1989, Ptacek and Travis 1996, Ptacek and Travis 1997, Ptacek 1998, Parzefall 2001, Plath et al. 2004). Similar scales of variation are also evident in the constraints on female mate choice versus male mate guarding or coercion (e.g., Farr 1989, Ptacek 1998, Plath et al. 2003, Plath et al. 2004). Variation in mating behavior related to male body size, for example, has been documented in Poecilia latipinna, one of a few molly species in which males are characterized by an enlarged dorsal fin (“sailfin”; Farr 1989, Travis 1994). Larger males show high rates of courtship and low rates of forced copulation attempts whereas smaller males exhibit the reverse. Among populations, however, males of similar size vary in courtship display rates (Ptacek and Travis 1996). Associated with this variation in male mating behavior is a female preference for larger males (Schlupp et al. 1994, Ptacek and Travis 1997) and, in some populations, for local versus foreign males (Ptacek and Travis 1997). Preference for larger males has also been demonstrated in Poecilia mexicana, one of several molly species characterized by an unexaggerated dorsal fin (“shortfin”) and inconspicuous courtship behaviors relative to the sailfin molly (Ptacek 1998, Plath et al. 2004). ____________ a
E-mail:
[email protected] Department of Biology, Elizabeth City State University, Elizabeth City, NC 27909 cDepartment of Ecology and Evolution, Princeton University, Princeton, NJ 08544 b
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In many populations, harassment by males is expected to be high, thus limiting the potential role of female-decision making in reproductive success (Farr 1989). In other populations, however, such as those found in caves, male aggression is low and females appear to exercise choice (Parzefall 2001, Plath et al. 2003, Plath et al. 2004). In addition, while apathy among females was high, in a laboratory study using non-cave P. mexicana, females disproportionately associated with sailfin P. latipinna males versus P. orri males, a shortfin species with a comparatively limited mating behavioral repertoire (Ptacek 1998). P. orri females also preferred the heterospecific with the larger dorsal fin. Jordan et al. (2005), using a single shortfin species, P. mexicana, also demonstrated the potential for female preference for an enlarged dorsal fin using conspecific-shaped models with manipulated fin dimensions. This diversity in male and female mating behavior within a single group of fish provides opportunities for testing hypotheses regarding environment, patterns of population divergence, and mating. In this study, we wanted to explore the possible interaction of environment with female preference in the divergence of a sexual selection linked trait in a shortfin molly. We tested the hypothesis that males and females would exhibit preferential associative behavior toward members of their local population versus conspecifics from a foreign population. We also tested the hypothesis that morphological distinctions between the two populations can be made. METHODS AND MATERIALS Sixty Poecilia gilli (Kner) were collected by seining each site during a single day in the last week of July 2003 from two streams in the Panama City, Panama area (9˚S-79.5˚W). Twenty-nine and thirty-one individuals, respectively, were collected from Juan Grande Ravine in the Gamboa Rainforest of Soberania National Park (hereafter Gamboa), and the Cardenas River in the Ciudad de Saber (hereafter Cardenas). The streams were separated by 16 km in linear distance with each separately flowing into the Panama Canal; thus, mixing of the two populations is unlikely. On site, we measured total length, maximum body depth, basal dorsal fin width, anterior dorsal fin height, posterior dorsal fin height, and basal caudal fin width. To determine which morphometric variables were most likely to define differences between the populations, we conducted discriminant function analysis using location as the grouping variable. Results from this analysis plus what is known about selection in mollies led us to conduct a post-hoc analysis of covariance (ANCOVA) to examine differences in dorsal fin height more thoroughly. The model included anterior dorsal fin height as the dependent variable, location and sex as the factors, and total length as the covariate. Equality of slopes was determined by including interactions of the covariate and main effects in the full model. Statistical procedures were conducted using Minitab Release 13.32. Fish were kept in aerated containers until shipped to the United States. There they were collectively housed in 90-liter species-specific tanks maintained at 26oC, pH 7.6. Fish were left undisturbed except for occasional tank maintenance and twice-daily feedings for six months prior to testing. The protocol for the behavioral experiments was similar to that used by Ptacek (1998). The test tank (75 x 25 x 30 cm) was partitioned with plexiglass into three compartments—two side compartments each 19 cm in length and a middle compartment that occupied the remaining volume at 37 cm in length. We conducted the following three experiments: (1) females were given a chance to preferentially associate with either a local male (from her population) or a
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foreign male (from the other population), (2) females were given a choice between two local females and two foreign females, and (3) males were given a choice of a local female and a foreign female. Seven focal fish from each population were used in all three experiments. Stimulus males and females were visually size-matched. Only females that had been isolated from males for six months, thus unlikely to be pregnant, were used. Pairs of stimulus females were used in the female-only experiments because the solitary focal females tended to shelter on the tank bottom in the absence of male fish. Males initiated movement more quickly than females, and after one fish became active, others did as well. Males were not paired because of their tendency for aggression. To avoid pseudoreplication, all stimulus combinations (i.e., combinations of individuals used in the side compartments) were unique even though fish were used more than once. Because poeciliids have demonstrated individual recognition (Witte and Massmann 2003, Dugatkin and Alfieri 1991), stimulus and focal fish, that were to be used in the same trial, were housed separately and out of view of each other. Trials were viewed for 10 minutes after the focal fish had visited both stimulus individuals. Because the data were not normally distributed, associative choice was determined by comparing ratios of time spent associating with each treatment versus total time using the Wilcoxon signed-rank test. Because we conducted multiple tests, we used α < 0.0169 as our acceptance criteria (sequential Bonferonni: Sokal and Rohlf 1997). The time that a test fish swam with snout pressed against a partition towards a treatment was deemed as time spent associating. Use of time spent has been shown to be a predictor of choice in other experiments involving poeciliids (see Houde 1997 for examples). To examine within- and among-observer reliability, some trials were videotaped and reviewed by the same observer, and others were viewed simultaneously by up to four observers. Reliability was measured as percent agreement on with which treatment the test fish spent the most time. RESULTS AND DISCUSSION The discriminant analysis was able to correctly group 87.5% of the fish, suggesting morphological distinction between the groups. Parameters with the largest coefficients and therefore most likely to be the greatest indicators of group separation were anterior dorsal fin height and total length. The full model ANCOVA indicated no interaction of total length with site or sex. In the main effects model, site was a significant factor (F = 31.74, P < 0.001) indicating that, with length controlled, anterior dorsal fin height was greater at Cardenas than Gamboa. Sex of fish was not significant. In the choice experiments, individuals spent at least 30 seconds associating. Females could discriminate between local and foreign males, preferentially associating with local males (Table 1; Fig. 1). It is unclear whether males or females could discriminate between local and foreign females as no preferential association with females was demonstrated. Based on previous choice experiments with mollies (Jordan et al. in press), we expected the effect that we were trying to detect to be relatively large, on the order of 20% proportional time difference. Assuming this effect size and n = 14, we calculated our statistical power to be 65% (methods obtained from Thomas and Juanes 1996). Within-observer reliability was 95%; among-observer reliability was slightly lower at 89%.
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Table 1. Results of Wilcoxon signed-rank tests from choice experiments. Left of the arrow indicates sex of test fish. * indicates rejection of the null hypothesis of no consistent choice. Experiment N Test Statistic P FemalesÆ Males 14 14 0.0134* FemalesÆ Females 14 59 0.7148 MalesÆ Females 14 50 0.9032
a. Fem ale choice of m ales 1 0.6
Difference in Proportion of Time Spent Associating
0.2 -0.2 -0.6 -1
b. Fem ale choice of fem ales
1.4 1 0.6 0.2 -0.2 -0.6 -1
c. Male choice of fem ales 1 0.6 0.2 -0.2 -0.6 -1 1
2
3
4
5
6
7
8
9
10
11 12 13 14
Test Fish Figure 1. Difference in proportion of time associating by individual in the three A positive value indicates choice of the local stimulus fish.
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experiments.
We found that male dorsal fin height, controlled for fish length, differed between locations. A number of factors could be responsible for this divergence. Habitat differences, such as flow, may select for larger dorsal fin in both sexes in the Cardenas river site. Males and females did not differ in dorsal fin height. A larger fin could also reflect higher food availability or quality. Sun exposure was higher at and near the Cardenas site, possibly resulting in greater productivity than in the more shaded Gamboa stream. Fin height also features prominently in mating systems among mollies. For example, a major taxonomic distinction has been drawn between the sailfin and short-fin molly (Ptacek and Breden 1998), thus raising the possibility that the dissimilarity is linked to differences in mating behavior. Spatial variation in male mating behavior has been demonstrated in the sailfin molly P. latipinna (Ptacek and Travis 1996) and in P. mexicana (Plath et al. 2003). Although forced copulation rather than courtship appears to dominate the reproductive behavior of shortfin molly males in the field (Farr 1989), some research has demonstrated the potential for male courtship behavior in this group (Balsano 1985). Female choice has been documented in cave-dwelling Poecilia mexicana populations where male aggression is low (Plath et al. 2003, Plath et al. 2004). In these populations, it is presumed that female choice has the potential to affect mating outcomes and perhaps subsequent male trait evolution. An enlarged dorsal fin might be favored where it enhances signaling but does not engender excessive risk such as to predation. A habitat with low visibility might present these conditions. Further testing is needed to connect dorsal fin height to differences in mating behavior related to the visual environment. Previous study has suggested that in some species, female shortfin mollies have a latent preference for an enlarged dorsal fin (Ptacek 1998, Gould et al. 1999, Jordan et al. 2005). Although it has not been demonstrated that females are able to exercise preference in the field, hence the modifier “latent,” this lack of evidence does not preclude the possibility that females are able to exercise mating choices. The latent preference for an enlarged dorsal fin hypothesis leads us to predict that females from both our study populations would associate disproportionately with the Cardenas males. Our data, however, do not support this prediction. This latent preference, however, may still exist in the females of the shorter-finned population but may be secondary to the preference for individuals from their own location. Studies using males from within a single population or with males from different populations exclusive of native males would help clarify this finding. Our results are consistent with other studies demonstrating choice for local versus foreign males in poeciliids (Ptacek and Travis 1997, Endler and Houde 1995) and in other fishes (Knight and Turner 2004, Wong et al. 2004). Such a bias for local males could facilitate trait divergence, whether that trait arose from a seemingly latent or overt mating preference or from environmental selective forces. ACKNOWLEDGMENTS We thank the Smithsonian Tropical Research Institute, Eldredge Bermingham and students, and Margaret Ptacek for advice and support during field collection. In addition, we thank two anonymous reviewers for their improvements to this manuscript. LITERATURE CITED Balsano, J.S., E.J. Randle, E.M. Rausch, and P. J. Monaco. 1985. Reproductive behavior and maintenance of all-female Poecilia. Environmental Biology of Fishes 12: 251-263. Dugatkin, L.A. and M. Alfieri. 1991. Guppies and the tit for tat strategy: preference based on past interaction. Behavioral Ecology and Sociobiology 28: 243-246.
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Endler, J.A., and A.E. Houde. 1995. Geographic variation in female preferences for male traits in Poecilia reticulata. Evolution 49: 456-468. Farr, J.A. 1989. Sexual selection and secondary sexual differentiation in Poeciliids: determinants of male mating success and the evolution of female choice. pp 91-124. In Meffe G.R. and F.F. Snelson (eds) Ecology and Evolution of Livebearing Fishes (Poeciliidae). Prentice Hall: Englewood Cliffs, NJ. Gould, J.L., T.J. Zabka, R.W. Malizia, A. Park, and J. Mukerji. 1999. Possible decision-making preadaptations in the molly. Animal Cognition 2: 91-95. Houde, A.E. 1997. Sex, Color, and Mate Choice in Guppies. Princeton University Press, Princeton, NJ. Jordan, R.C., D.V. Howe, R.T. Knight, and J.L. Gould. 2005. Female choice linked to male dorsal fin height in a shortfin molly. Journal of Ethology (in press). Knight, M.E. and G.F. Turner. 2004. Laboratory mating trials indicate incipient speciation by sexual selection among populations of the cichlid fish Pseudotropheus zebra from Lake Malawi. Proceedings of the Royal Society of London B 271: 675-680. Minitab Inc. 2000. Minitab Release 13.32 Minitab Inc, State College, PA. Parzefall, J. 2001. A review of morphological and behavioural changed in the cave molly, Poecilia mexicana, from Tabasco, Mexico. Environmental Biology of Fishes 62:263-275. Plath M., J. Parzefall, and I. Schlupp. 2003. The role of sexual harassment in cave and surface dwelling populations of the Atlantic molly, Poecilia mexicana (Poeciliidae, Teleostei) Behavioral Ecology and Sociobiology 54:303-309. Plath M., J. Parzefall, and K.E. Körner. 2004. Sexual selection in darkness? Female mating preferences in surface- and cave-dwelling Atlantic mollies, Poecilia mexicana (Poeciliidae, Teleostei) Behavioral Ecology and Sociobiology 55:596-601. Ptacek, M.B. 1998. Interspecific mate choice in sailfin and shortfin species of mollies. Animal Behviour 56: 1145-1154. Ptacek, M.B. and F. Breden. 1998. Phylogenetic relationships among the mollies (Poeciliidae: Poecilia: Mollienesia group) based on mitochondrial DNA sequences. Journal of Fish Biology 53 (Supplement A): 64-81. Ptacek, M.B. and J. Travis. 1996. Inter-population variation in male mating behaviours in the sailfin mollie, Poecilia latipinna. Animal Behaviour 52: 59-71. Ptacek, M.B. and J. Travis. 1997. Mate choice in the sailfin molly, Poecilia latipinna. Evolution 51: 1217-1231. Schlupp, I., C.A. Marler, and M.J. Ryan. 1994. Benefit to male sailfin mollies of mating with heterospecific females. Science 263:373-374. Schlupp I., R. McKnab, and M.J. Ryan. 2001. Sexual harassment as a cost for molly females: bigger males cost less. Behaviour 138:277-286. Sokal R.R. and F.J. Rohlf. 1997. Biometry. WH Freeman and Co, New York, NY. Thomas, L. and F. Juanes. 1996. The importance of statistical power analysis: an example from Animal Behaviour. Animal Behaviour 52:856-859. Travis, J. 1994. Ecological genetics of life-history traits: variation and its evolutionary significance. pp171-204. In: L.A. Real (ed) Ecological Genetics. Princeton University Press: Princeton, NJ Witte K, and R. Massmann. 2003. Female sailfin mollies, Poecilia latipinna, remember males and copy the choice of others after one day. Animal Behaviour 65:1151-1159. Wong, B.B.M., J.S. Keogh, and M.D. Jennions. 2004. Mate recognition in a freshwater fish: geographical distance, genetic differentiation, and variation in female preference for local over foreign males. Journal of Evolutionary Biology 17: 701-708.
Author Final Copy to: Journal of Freshwater Ecology
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