Behav Ecol Sociobiol (2004) 55:461468 DOI 10.

1007/s00265-003-0727-8

ORIGINAL ARTICLE

Lars B. Pettersson Indar W. Ramnarine S. Anette Becher Rajindra Mahabir Anne E. Magurran

Sex ratio dynamics and fluctuating selection pressures in natural populations of the Trinidadian guppy, Poecilia reticulata
Received: 5 June 2003 / Revised: 20 August 2003 / Accepted: 28 October 2003 / Published online: 10 December 2003  Springer-Verlag 2003

Abstract In many species, population sex ratios have farreaching consequences for a wide variety of populationlevel and behavioural processes and can directly influence sexual selection through differential effects on male and female mating behaviour. Although sex ratios are often treated as more or less stable population characteristics, recent theoretical evidence suggests that sex ratios fluctuate under many conditions, and that the amplitude of these fluctuations can be considerable. Few studies have attempted to quantify this variation in systems with prominent, sex ratio-dependent sexual conflict. One of the species with the greatest potential to integrate these factors in the wild is the Trinidadian guppy, Poecilia reticulata. In this study, we quantified natural sex ratio variation both as detailed longitudinal studies of focal guppy populations and as snapshot estimates across a range of freshwater habitats. In line with theoretical predictions, we expected to detect significant sex ratio variation over time. We also investigated the association between juvenile and adult sex ratios to quantify a possible compensatory feedback implied in standard models of sex ratio evolution. Our results confirm that population-level sex ratios in wild guppy populations have a range of dynamic features, with all four focal populations showing significant variation in sex ratio over time. The survey showed that juveniles were generally close to equal (50:50) sex ratios whereas 7 out of 11 adult
Communicated by J. Krause L. B. Pettersson ()) Department of Ecology, Lund University, Ecology Building, SE-223 62 Lund, Sweden e-mail: lars.pettersson@zooekol.lu.se L. B. Pettersson S. A. Becher A. E. Magurran Gatty Marine Laboratory, School of Biology, University of St Andrews, St Andrews, Fife, KY16 8LB, Scotland, UK I. W. Ramnarine R. Mahabir Department of Life Sciences, University of the West Indies, St. Augustine, Trinidad & Tobago

sex ratios differed significantly from equality. We found no evidence that a surplus of juveniles of the locally rarer sex had been produced. The results indicate that sex ratios and hence the balance between sexual selection and sexual coercion is normally fluctuating in nature, despite juvenile ratios being close to equality. Keywords Fish Population dynamics Sex ratio Sexual selection

Introduction
The sex ratio has emerged as a central concept in modern evolutionary ecology. Not only does it provide key links between sexual selection, population dynamics and evolutionary trajectories (e.g., Charnov 1982; Caswell and Weeks 1986; Magurran 2001; Pen and Weissing 2002), but it can also be the focus of adaptive allocation strategies where individuals adjust their relative investment in males and females to match current and expected fitness payoffs (e.g., Trivers and Willard 1973; CluttonBrock 1986; Frank 1990; West and Sheldon 2002). These two aspects of the sex ratio, that focusing on populationlevel processes and that focusing on offspring sex allocation, have a central property in common. They are essentially dynamic processes (Caswell and Weeks 1986; Byholm et al. 2002). The basis for this is that when one sex dominates numerically, parents biasing their brood towards the rarer sex will experience a fitness advantage, something that can take place even when production costs differ (Fisher 1958; Trivers and Willard 1973). This feedback process is expected to lead to convergence on stable sex ratios or at least to move the sex ratio in the direction of a stable situation (Fisher 1958; Caswell and Weeks 1986; Ranta et al. 2000). However, while traditional Fisherian (Fisher 1958) models of sex ratio dynamics focus on panmictic, homogenous populations where convergence is clearly predicted, a new generation of models (e.g., Caswell and Weeks 1986; Lindstrm and Kokko 1998; Ranta et al. 2000) has stressed that sex ratio

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dynamics, just like population dynamics, are inherently parameter sensitive and that convergence is just one of many possible outcomes. In many situations, stable oscillations, chaotic fluctuations or even population extinction can be the likely outcome when a sex ratio is perturbed (Caswell and Weeks 1986). In addition, spatial heterogeneity can further influence the stability and dynamics of sex ratios (Juillard 2000; Ranta et al. 2000; Pen and Weissing 2002). Hence, rather than homing in towards stability, there is good evidence that sex ratios may normally fluctuate under many conditions, and that the amplitude of these fluctuations can be considerable (Caswell and Weeks 1986; Lindstrm and Kokko 1998; Ranta et al. 2000). Interestingly, changes in sex ratios are known to affect many aspects of male and female behaviour (CluttonBrock and Vincent 1991; Krupa and Sih 1993; Magurran and Seghers 1994a; Clutton-Brock et al. 1997; Croft et al. 2003). In fish, features such as male mating tactics, female choice, and sperm production characteristics can be affected (Kvarnemo and Ahnesj 1996; Balshine-Earn 1996; Evans and Magurran 1999; Jirotkul 1999a). But while the interaction between behavioural flexibility and sex ratios is just beginning to be explored (e.g., Kvarnemo and Ahnesj 1996; Evans and Magurran 1999; Jirotkul 1999a; Croft et al. 2003), very little is known about how sex ratios shaping such interactions vary under natural conditions, how consistent they are between populations and how environmental factors influence the resulting population sex ratios. One of the species with the greatest potential to integrate variation in mating behaviour with sex ratio dynamics in the wild is the guppy, Poecilia reticulata, a poeciliid fish native to Trinidad. It is widely distributed across the island and is found in virtually every freshwater habitat (Magurran and Phillip 2001; Magurran 2001). Guppies have a promiscuous mating system (Houde 1997) with a central feature being female choice for colourful males (Houde and Endler 1990; Houde 1997). In contrast, males switch between two different mating strategies, either courting females using sigmoid display behaviour (Houde 1997) or engaging in forced mating attempts (sneaking), thereby potentially overriding female choice (Magurran 1998; 2001). The balance between courting and sneaking is affected by multiple factors (reviewed in Magurran 2001) and increasing evidence suggests that sex ratios, either seen as operational sex ratios (i.e., the ratio of sexually active males to the sum of sexually active males and receptive females; CluttonBrock and Vincent 1991; Jirotkul 1999a, 2000) or as general sex ratios (Evans and Magurran 1999), is one of the most important. For instance, Evans and Magurran (1999) demonstrated that males experiencing male-biased (high) sex ratios during maturation had higher sneaking rates than males that developed under even sex ratios. In contrast, evidence for a genetic basis of differences in male mating behaviour is limited, despite thorough investigations (cf. Rodd and Sokolowski 1995). Unfortunately, despite being a central population characteristic

with potentially far-reaching consequences, there has been surprisingly little quantitative evidence for temporal sex ratio variation in wild guppy populations (but see Seghers 1973; Rodd and Reznick 1997), yet a temporal understanding of sex ratios seems more and more essential (Caswell and Weeks 1986; Lindstrm and Kokko 1998; Palmer 2000; Ranta et al. 2000). Further, as models of sex ratio dynamics predict a feedback between population-level and brood-level sex ratios (Fisher 1958; Caswell and Weeks 1986; Ranta et al. 2000), there is a need to relate juvenile sex ratios to adult ones across populations. To avoid influences from plastic sex determination (cf. Baroiller and DCotta 2001), such studies need to be carried out in the wild. The aim of the present study is to address sex ratio variation in wild guppy populations, both as snapshot estimates of the association between sex ratios of newborn and adult guppies across several guppy populations from contrasting environmental conditions, and as longitudinal studies of sex ratio variation in four focal populations. By explicitly including spatial and temporal aspects, we seek to avoid drawbacks associated with less structured designs, such as pattern detection difficulties and overgeneralization (cf. Palmer 2000; Byholm et al. 2002). In line with Caswell and Weeks (1986), we expect significant sex ratio variation over time within populations. Further, a negative association between juvenile and adult sex ratios within populations would indicate the presence of a dynamic feedback such as the ones implied in traditional Fisherian (Fisher 1958) models of sex ratio dynamics.

Methods
Study sites and experimental populations We selected 11 guppy populations throughout Trinidad for initial quantification of juvenile and adult sex ratios (Fig. 1, Table 1). Sampling took place in April 2000 and the populations surveyed represented four major drainages, headstream as well as downstream sites, contrasting predation pressures, and considerable differences in water quality (Table 1). Our sampling strategy was based on the fact that ecological factors vary substantially between guppy populations (e.g., Liley and Seghers 1975; Magurran and Phillip 2001) and that the use of replicated pairs of headstream and downstream sites provides means for quantifying parallellism in ecological and evolutionary patterns between streams (e.g., Magurran 2001; Reznick et al. 2001). Predators are excluded from headstreams by rapids or waterfalls, yielding low-predation communities (Magurran and Phillip 2001). Guppies in these isolated streams suffer only minor predation on juveniles from the omnivore Harts rivulus, Rivulus hartii (Carvalho et al. 1996). In contrast, downstream fish live in high-predation communities where predation from the pike cichlid, Crenicichla alta, and other species of cichlids, erythrinids, and characins (Carvalho et al. 1996), is primarily directed at large, mature individuals. Based on the 11 populations surveyed, two focal population pairs (Upper Tunapuna and Caura; Upper Aripo and Lower Aripo) were then selected for monthly sampling of sex ratios during 1 year. Climatological data for the period were obtained from the Meteorological Service, Piarco International Airport, Trinidad.

463 Fig. 1 Location of the sampling sites on Trinidad. Shading indicates altitudes >100 m above sea level

Table 1 Sampling sites for populations of the Trinidadian guppy, Poecilia reticulata and their associated grid references, geographical drainage systems, predation status, water quality, and number of adult guppies collected for initial sex ratio estimates. Population names are abbreviated as follows: U upper; M mid-; L lower. Water quality is denoted as +++: clear and clean; ++: moderately clean; +: polluted and/or disturbed

Grid reference U Aripo L Aripo U Pilote L Pilote U Tunapuna Caura (M Tacarigua) L Tacarigua U Oropouche L Oropouche Pitch Lake Silver Stream PS 931 817 PS 938 786 QS 042 188 QS 116 218 PS 759 797 PS 787 804 PS 749 772 QS 024 852 QS 043 788 PS 503 316 PS 551 299

Drainage Caroni Caroni Pilote Pilote Caroni Caroni Caroni Oropouche Oropouche La Brea La Brea

Predation status Low High High High Low High High Low High High High

Water quality +++ ++ +++ + +++ +++ + +++ +++ +a ++

n 75 93 102 109 91 128 162 98 149 76 81

a Pools are clear, but subject to natural oil seepage and extreme water temperatures (reaching as high as +41C; Kenny 1995)

Sampling procedures The protocol for the initial sex ratio survey consisted of randomly collecting approximately 100 adult and at least 30 juvenile guppies from each population using dip nets and seine nets. The section of stream sampled at each site was short enough to be fished thoroughly. On average, the length of stream used was 510 m. In upstream localities such as the Upper Aripo and Upper Tunapuna, this typically represented two to three pools connected by riffles, whereas in downstream sites, a corresponding section of the stream was sampled using the same techniques. Adult guppies were sexed using external characteristics (Houde 1997) and were then released back into the stream. Juveniles were stored in 70% ethanol until further processing. The focal populations were then sampled monthly by two of the authors (I.W.R. and R.M.). Using the same protocol as in the survey, approximately 100 adult individuals were collected at each of the four sites, sexed, and were then released back into the stream. The time taken to collect this number of fish was noted. If fewer

than 100 fish were present, the time after which no additional fish were caught was noted. This approach allowed catch per unit effort to be measured. Histological analysis We used histological techniques to sex the collected guppy juveniles. Although time consuming, histological sex characterisation allows reliable identification of sex in newborn guppies (Takahashi 1975) and avoids influences of environmental sex determination that could affect results when quantifying the sex ratio by rearing juveniles to adulthood (Baroiller and DCotta 2001). The processing of collected juveniles began by measuring their length, transferring them individually to Bouins fixative (Hinton 1990), and then dehydrating them through an alcohol series. After dehydration, fish were transferred to benzene before being embedded in paraffin wax. Serial transverse sections (7 m) covering the gonad were stained with eosin and used for sexing

464 under the microscope. Sex identification followed Takahashi (1975). Presence of oocytes was used as the criterion for females whereas presence of testes was used as the criterion for males (Takahashi 1975; Billard 1986). All 330 individuals investigated could be unequivocally sexed by one of the authors (L.B.P.). The sex identification was confirmed through a re-analysis of a random subset (66 individuals, blind analysis) of the data. Statistical procedures Differences from equal (50:50) sex ratios in both the initial survey and the longitudinal monitoring of the focal populations were evaluated as 95% confidence intervals for the estimated binomial parameters (Zar 1999; Wilson and Hardy 2002). Furthermore, temporal heterogeneities across and within focal populations were evaluated using log-linear models, assuming appropriate error distributions and link functions (Wilson and Hardy 2002). Quantification of juvenile and adult sex ratios in the initial survey was performed using SAS Proc GENMOD (SAS Institute 1999) assuming binomial error distribution and a logit link function. Size differences between juveniles from different populations were evaluated using analysis of variance (ANOVA). Catch per unit effort (CPUE) for each of the focal populations was calculated as the number of guppies captured per sampling divided with time spent collecting them (cf. Perrow et al. 1996). The relation between this measure of population size and two potentially influential factors (rainfall, and position within river), was evaluated with a repeated measures model using SAS Proc MIXED (SAS Institute 1999). When necessary to meet model assumptions, data were transformed according to standard practise (e.g., Zar 1999). Finally, in addition to estimating 95% confidence intervals for the binomial parameters, we analysed the temporal sex ratio variability in the focal populations using SAS Proc CATMOD (SAS Institute 1999). This analysis was carried out in a two-step process. First, an overall test on the pooled data set was conducted in order to assess general heterogeneity within the sampled populations (cf. Wilson and Hardy 2002). Having identified such patterns, we then proceeded to assess variability on a population-level basis using the same methodology.

Results
Sex ratio survey There were significant sex ratio differences between populations (log-linear analysis, population: c2=32.07, df=10, P=0.0004; Fig. 2), and the mean sex ratio of adults was different from the mean juvenile sex ratio (log-linear analysis, age: c2=10.12, df=1, P=0.0015; Fig. 2). Further, the relation between juvenile and adult sex ratios differed between populations, as indicated by a significant interaction term (log-linear analysis, population age: c2=31.00, df=10, P=0.0006; Fig. 2). All juvenile sex ratios were close to equal (Fig. 2) and there was no evidence of a negative correlation between adult and juvenile sex ratios (Fig. 2). The sizes of the collected juveniles matched general patterns of size variation in juvenile guppies, with individuals from upstream sites being bigger (two-way ANOVA, population: F10,307=7.59, P<0.0001). There was no size difference between juvenile males and females (two-way ANOVA, sex: F1,307=0.96, P=0.33). Temporal variation in sex ratio The temporal distribution of sex ratios was significantly heterogeneous (log-linear analysis, likelihood ratio=66.40 df=11, P<0.0001; Fig. 3). When proceeding to analyse the same patterns within populations, all four sampled populations proved to be significantly variable over time (log-linear analysis, Upper Tunapuna: likelihood ratio=22.04 df=11, P<0.024; Caura: likelihood ratio=73.16 df=11, P<0001; Upper Aripo: likelihood ratio=77.19, df=11, P<0.0001; Lower Aripo: likelihood ratio=46.93 df=11, P<0.0001; Fig. 3). Further analysis using confidence intervals showed that about a fourth of the observations differed significantly from an even sex ratio (Fig. 3).

Fig. 2 The mean juvenile and adult sex ratios ( 95% CI) of the populations of the Trinidadian guppy, Poecilia reticulata surveyed in the April 2000 sampling. Juvenile sex ratios were estimated using n=30 from each population, sample sizes for adults were n=75162 from each population (Table 1); * significant deviations from equal sex ratios

465 Fig. 3 The mean adult sex ratio (95% CI) per month of the focal populations. Upper Tunapuna and Caura belong to the Tacarigua river, while Upper and Lower Aripo belong to the Aripo river. Upper Tunapuna and Upper Aripo are headstream sites, while Caura and Lower Aripo are downstream sites. Sample sizes for the different populations were: Upper Tunapuna, n=12116, mean n (n)=82; Caura, n=29135, n=105; Upper Aripo, n=75126, n=111; Lower Aripo, n=93 131, n=107; * significant devia tions from equal sex ratios

Analysis of population densities showed that overall densities, as measured by catch per unit effort, were higher in the two downstream populations than in the headstream populations (Caura and Lower Aripo: CPUE=3.230.49 [meanSE]; Upper Tunapuna and Upper Aripo: CPUE=6.700.70; MANOVA, F1,19.5=6.79, P=0.017). Monthly rainfall did not have an overall effect on population density (MANOVA, F1,20.4=1.20, P=0.28). However, there was a trend towards rainfall affecting headstream populations differently from downstream populations (MANOVA, F1,20.4=3.28, P=0.08).

Discussion
Our results demonstrate that population-level sex ratios in wild guppy populations have a range of dynamic features, with juveniles generally being close to equal (50:50) sex ratios whereas 7 out of 11 adult sex ratios differed significantly from equality (Fig. 2). The survey, producing a snapshot estimate of sex ratios, found no evidence for compensating processes (cf. Fisher 1958) in which a surplus of juveniles of the locally rarer sex had been produced. When viewed over time, all four focal populations show significant variation in sex ratio. This clearly stresses the need to put local sex ratios and the consequences they have for sexual selection into a dynamic perspective (cf. Magurran 2001). Rather than converging on stable sex ratios, our populations appear to be undergoing long-term oscillations consistent with predictions by recent models of sex ratio dynamics (e.g., Caswell and Weeks 1986; Lindstrm and Kokko 1998; Ranta et al. 2000; Pen and Weissing 2002). The prevalence of this pattern has not been recognised before, but is likely to influence a range of features in systems where, like the Trinidadian guppy, sexual selection is

strongly affected by the local sex ratio (Evans and Magurran 1999; Jirotkul 1999a; Jirotkul 2000). Our finding that juvenile sex ratios were close to equal whilst adult sex ratios from the same population deviated strongly from equality (Fig. 2) may indicate that juvenile sex allocation is influenced by long-term averages rather than current local sex ratios. Despite fluctuating considerably, our four focal populations nevertheless gave longterm averages close to equal sex ratios (Fig. 2). What can then be causing the significant deviations in adult sex ratios? Rodd and Reznick (1997) suggested that differential male mortality (cf. Seghers 1973; Liley and Seghers 1975; Reznick et al. 1996) creates these patterns. Males, being colourful, small and active, have been suggested to face relatively higher predation pressure than females at some sites, in particular in headstream populations (Seghers 1973; Liley and Seghers 1975; Rodd and Reznick 1997). In a study of eight populations, Rodd and Reznick (1997) found 90% of all observations within a pooled dataset to be female biased, an observation attributed to differential mortality. However, our results show both male and female biased populations, with a far lower share of female biased sites (Fig. 2). When adding the dynamic aspect with monthly samplings, our results show that the same populations may go from being male biased to female biased over the season. Hence, if differential mortality is the only factor underlying these patterns, then mortality rates of males and females will need to change over the year. Alternatively, other factors could affect adult sex ratios. To investigate this possibility, we identified a number of general differences between sites (Table 1). There were no significant correlations between sex ratio and these parameters (results not shown), but one important exception did emerge. Pitch Lake, having the surveys most skewed sex ratio (Fig. 2) is a site with natural oil seepage and extreme temperatures (Agard et al. 1993; Kenny 1995). Male guppies are more

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sensitive to high temperatures than females (Gibson 1954), which most likely causes high male mortality at this site. The dynamics at this location still remain to be investigated. To conclude, given that these general differences between sites were unable to explain the seasonal sex ratio dynamics, this asks for an alternative explanation. Swimming activity and performance in relation to peak water velocity could be a possibility (cf. Crow 1981; Nicoletto 1996; Nicoletto and KodricBrown 1999; Croft et al. 2003). Movement decisions of individuals within and between patches can be sex ratio dependent (Lawrence 1987; Krupa and Sih 1993) and may contribute to sex ratio fluctuations between individual pools (Jirotkul 1999a; Croft et al. 2003). On a more general scale, recent evidence suggests that there are inter-population differences in swimming ability between males from different populations (Nicoletto and KodricBrown 1999). Males from headstream sites had significantly higher swimming performance than males from downstream sites (Nicoletto and Kodric-Brown 1999). Given that maximum stream velocity is most likely highest at headstream sites, this indicates that the ability to cope with peak flow may be a key trait in headstream populations. Corresponding studies of female swimming ability have not been carried out, but theoretical evidence (Webb 1975) suggests that females should be able to cope with higher water velocities than males. This sex-specific and potentially population-specific difference in ability to withstand high water velocity could then influence sex ratios in similar ways as differential mortality. Interestingly, there is evidence that as many as 5% of adults and juveniles move away from local populations over a 12 day period (Reznick et al. 1996). This corresponds to a 25% turnover of the local population in 6 weeks. Hence, a considerable number of fish are moving between subpopulations and could be subject to being swept downstream (cf. Shaw et al. 1992; Becher and Magurran 2000). In line with this, the present study found a tendency for peak rainfall to affect population density differently in headstream and downstream populations. In other words, differential swimming ability and discharge variation can potentially affect sex ratio dynamics. The extent to which these factors influence sex ratios in the wild remains to be quantified. Sex ratios such as the range described in this study have important consequences for male mating tactics, female choice and sperm production characteristics. Jirotkul (1999a) found significant shifts in male mating tactics as the sex ratio varied between 0.17 and 0.83. When the test population was female-biased, individual males devoted a large proportion of time to courtship and engaged in many sigmoid displays. As the sex ratio became progressively more male-biased, females exerted a stronger preference for orange coloration, possibly because the costs of mate choice were lower. With the exception of extreme male bias (five males and a single female in the arena: SR=0.83), where 60% of matings followed sneak attempts, all copulations were preceded by sigmoid displays. Jirotkul (2000) subsequently showed

that the opportunity for sexual selection increased as the sex ratio became biased towards males. She attributed this to the increase in both male-male competition (Jirotkul 1999b) and female choice (Jirotkul 1999a). In Jirotkuls work all males and females were sexually receptive and her results therefore demonstrate how changes in operational sex ratio mediate reproductive interactions (Clutton-Brock and Vincent 1991; Kvarnemo and Ahnesj 1996). Nonetheless, in the wild, only a small fraction of females are receptive at any one time (Magurran and Seghers 1994b). Evans and Magurran (1999) examined the mating behaviour of males reared in male-biased (11 males : 5 females), female-biased (5 males : 8 females) and evenly-balanced (8 males : 8 females) groups. Males were tested both in their rearing groups and in a 1:1 sex ratio; females were non-receptive. They observed the same pattern as Jirotkul: males from the male-biased rearing treatment performed relatively more sneaky mating attempts and relatively fewer sigmoid displays. The increased incidence of sneaky copulations persisted when fish were tested 24 h later in a 1:1 sex ratio, indicating that mating strategies remain fixed for short periods at least. In the wild, this means that the sex ratio variability over time described in the present study is likely to interact with the possibilities for female choice and alternative male mating behaviours. Magurran (2001) argued that the balance between sexual selection and sexual coercion varies across populations in Trinidad. In low predation populations, females have more opportunities to express choice, whereas in high predation localities preoccupation with predator avoidance by females, and greater use of sneaky tactics by males, means that sexual coercion may predominate. However, as this study has demonstrated, sex ratios are far from stable in wild guppy populations. Despite equal sex ratios at birth, adult sex ratios undergo marked variation over the year, stressing that external processes such as predation and other environmental influences can contribute substantially to sex ratio fluctuations. Given the importance of local sex ratio for the balance between sexual selection and sexual coercion, this dynamic process is likely to have far-reaching consequences for the evolutionary outcome of sexual selection in wild populations. Integrating physiological performance and local adaptation with these processes is urgently needed.
Acknowledgements We thank the Meteorological Services, Piarco International Airport, Trinidad and Tobago for providing meteorological data, Joakim Larsson for histological advice and Jill McVee for help processing samples. This study was supported by the Hellmuth Hertz Foundation (L.B.P.), The Swedish Foundation for International Cooperation in Research and Higher Education (L.B.P.) and the Natural Environment Research Council UK (S.A.B. and A.E.M.)

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